WO2010126073A1 - Expression cassette for secretion/expression of polypeptide, and use thereof - Google Patents

Abstract

Disclosed are a method for the efficient secretion/expression of a polypeptide by means of a microorganism, preferably a bacterium belonging to the genus Bifidobacterium and a means for the method. Specifically disclosed is an expression cassette characterized by comprising: (i) a promoter selected from the group consisting of a promoter for a cbaH gene derived from a bacterium belonging to the genus Bifidobacterium, a promoter for rplJ gene derived from a bacterium belonging to the genus Bifidobacterium, and a promoter for hup gene derived from Bifidobacterium breve; (ii) DNA encoding a signal sequence selected from the group consisting of a signal sequence for a product of BL1181 gene derived from a bacterium belonging to the genus Bifidobacterium, and a signal sequence for a product of amyB gene derived from a bacterium belonging to the genus Bifidobacterium; and (iii) DNA encoding a polypeptide or a cloning site into which the DNA is to be inserted.

Description

Expression cassette for the secretory expression of polypeptides and uses thereof

The present invention relates to secretory expression of a polypeptide in a microorganism. In particular, the present invention relates to the secretion of polypeptides in Gram positive bacteria such as Bifidobacterium used to deliver polypeptides to mucosa and / or tumor tissue for the purpose of treating or preventing diseases or disorders. It relates to means and methods for expression.

So far, the usefulness of various recombinant microorganisms has been studied for the purpose of treating or preventing diseases (Non-Patent Document 1). For example, for the purpose of delivery of useful polypeptides to the mucosa, methods using Lactococcus, Lactobacillus, Bifidobacterium, etc. have been studied (Non-Patent Documents 2, 3, 4). For the purpose of tumor therapy, methods using Clostridium, Salmonella or Bifidobacterium that express prodrug converting enzyme have been studied (Non-patent Documents 5, 6, and 7). . Furthermore, the delivery of RNAi using Escherichia coli is also examined (Non-patent Document 8). The method using a recombinant microorganism can overcome the problems of toxicity and blood half-life when an effective nucleic acid or polypeptide is administered systemically, and can be a means for effectively delivering the nucleic acid or polypeptide. Furthermore, in the process of production, a process that requires a large amount of cost such as purification can be omitted, and the utility can be provided easily and / or inexpensively as compared with the recombinant protein product.

Among these host microorganisms, Bifidobacterium is a non-pathogenic resident bacterium that is also used for probiotics and is an obligate anaerobic bacterium that can be used for delivery to both mucous membranes and tumors. Therefore, it is considered to have excellent characteristics from the viewpoint of safety, biological containment, and versatility.

In addition, Bifidobacterium is far more durable in the intestinal tract than L. ラ ク ト lactis, a non-symbiotic bacterium that has not been artificially improved for gastrointestinal resistance, The possibility of showing favorable pharmacokinetics for treatment and prevention of diseases has been suggested (Non-patent Document 9). Therefore, it is possible that Bifidobacterium is superior to recombinant L. に お い て lactis for the purpose of delivering useful polypeptides to the gastrointestinal mucosa for the treatment of inflammatory bowel disease and the like.

When Bifidobacterium is administered intravenously, it survives and grows only in tumors with very high selectivity (Non-patent Document 10). This is probably due to the fact that the tumor has a hypoxic region. Using this property, for the treatment of tumors, Escherichia coli cytosine deaminase (CD), which can convert the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), Bifidobacterium that is produced without secretion is produced using the promoter of the hup gene of B. longum, and tumor therapeutic effects using this bacterium have been studied (patent document) 1). Regarding this method, 5-FU produced by Bifidobacterium itself by enzyme CD acts not only on tumor cells but also on fungi, so that the therapeutic effect is limited by the suicide of the fungus. Considering this, 5-FU resistant bacteria have been obtained in vitro (Patent Document 2). However, in order to exert an antitumor effect in vivo, an active metabolite in which 5-FU is converted by cellular enzymes, 5-fluoro-UTP (5-FUTP) or 5-fluoro-dUMP (5-FdUMP) Is necessary (Non-patent Document 11). These are also toxic to bacteria and may still have limited efficacy. Moreover, this method requires administration of a large amount of 5-FC to a living body separately from Bifidobacterium, and is greatly limited. Therefore, the use of Bifidobacterium that secretes and expresses a useful polypeptide is also required as a more effective method in tumor therapy applications.

In general, there are many bottlenecks in the secretory expression of heterologous polypeptides, especially the biologically active polypeptides in bacteria, and secretion expression is difficult due to problems inherent in each heterologous polypeptide. In many cases. However, a great deal of research has been accumulated especially in Bacillus subtilis and Lactococcus lactis (Non-patent Documents 12, 13, and 14).

On the other hand, there are very few reports on secretory expression using Bifidobacterium compared with them. For example, in Bifidobacterium, the secretion expression of interleukin-10 (IL-10) has been attempted using the promoter of B. longum hup gene and the signal sequence of galactosidase. Has been reported to be as low as 22 pg / ml (Non-patent Document 15). Similarly, secretory expression of basic fibroblast growth factor (FGF-2) was attempted using the hup gene promoter, and the culture supernatant was precipitated with trichloroacetic acid and the protein was detected by Western blotting. Has been reported (Non-Patent Document 16). In addition, attempts have been reported to secrete and express foreign polypeptides using the promoter and signal sequence of the amyB gene of B. adolescentis (Non-patent Documents 17 and 18). Here, the polypeptide in the culture supernatant is detected by SDS-PAGE in an active form or in an ammonium sulfate precipitated form.

Furthermore, the expression of endostatin in Bifidobacterium using an E. coli promoter has been reported. In this report, a signal sequence is not used, and an expression protein is detected by Western blotting after purification from disrupted cells (Non-patent Document 19). Recently, TRAIL / Apo2-L has been expressed in the same expression system, and its effect has been shown in tumor-bearing mice. However, the expression level and specific activity of TRAIL / Apo2-L, and also how TRAIL / Apo2-L is expressed under the control of the E. coli promoter and acts outside the Bifidobacterium without a signal sequence Is not shown (Non-patent Document 20). Further, in Patent Document 1, although there is a suggestion about the expression of interleukin-2 (IL-2) and endostatin in B. longum, the actual expression thereof has not been reported so far.

In summary, Bifidobacterium is considered to be a host with superior characteristics that cannot be found in other treatment methods using recombinant bacteria that may provide unprecedented medical value. However, regarding the secretion expression of heterologous polypeptides, it was not always satisfactory.

Japanese Patent No. 3642755 International Publication WO2006 / 109619

Vassaux G, et al. J Pathol. 2006. 208 (2): 290-8. Remaut E, et al. Bioscience and Microflora. 2006. 25 (3): 81-97. Liu X, et al. Appl Environ Microbiol. 2008. 74 (15): 4626-35. Takata T, et al. J Gene Med. 2006. 8 (11): 1341-6. Wei MQ, et al. Cancer Lett. 2008. 259 (1): 16-27. Nemunaitis J, et al. Cancer Gene Ther. 2003. 10 (10): 737-44. Sasaki T, et al.Cancer Sci. 2006. 97 (7): 649-57. Xiang S, et al. Nat Biotechnol. 2006. 24 (6): 697-702. Watson D, et al. BMC Microbiol. 2008. 8: 176. Kimura NT, et al. Cancer Res. 1980. 40 (6): 2061-8. Erbs P, et al.Cancer Res.2000. 60 (14): 3813-22. Bolhuis A, et al. Appl Environ Microbiol. 1999. 65 (7): 2934-41. Li W, et al. Res Microbiol. 2004. 155 (8): 605-10. Morello E, et al. J Mol Microbiol Biotechnol. 2008. 14 (1-3): 48-58. Reyes Escogido ML, et al. Biotechnol Lett. 2007. 29 (8): 1249-53. Shkoporov AN, et al. Biotechnol Lett. 2008. 30 (11): 1983-8. Park MS, et al. Lait 2005. 85: 1-8. Moon GS, et al. Appl Environ Microbiol. 2005. 71 (9): 5630-2. Xu YF, et al. Cancer Gene Ther. 2007. 14 (2): 151-7. Hu B, et al. Cancer Gene Ther. 2009. 16 (8): 655-63.

The inventors of the present invention thought that by efficiently secreting and expressing a useful polypeptide in Bifidobacterium, a technology having a great utility value that has not been available in the past can be provided for medical and other uses. However, the promoter and signal sequences conventionally used in Bifidobacterium and combinations thereof have not always been satisfactory for secreting and expressing useful polypeptides.

Therefore, a first object of the present invention is to provide a method and means for efficiently secreting and expressing a useful polypeptide in a microorganism, preferably Bifidobacterium. The second is to provide a pharmaceutical composition comprising means for delivering useful polypeptides to mucosa and / or tumor tissue. A third object is to provide means and methods for the treatment and prevention of diseases or disorders by utilizing the pharmaceutical composition.

The present inventors have made extensive studies for the purpose of obtaining a combination of a promoter and a signal sequence capable of efficiently secreting and expressing a biologically active useful polypeptide in Bifidobacterium. As a result, the present inventors have found a combination of a promoter and a signal sequence capable of secreting and expressing a useful polypeptide having biological activity at a high level as compared with conventionally known expression cassettes. It was also found that when a DNA encoding about 1 to 20 amino acids after the signal sequence of a specific gene is inserted after the signal sequence, the secretory expression efficiency of the polypeptide is further increased. Furthermore, the present inventors have found in animal experiments that bifidobacteria into which an expression vector containing the expression cassette has been introduced show a long-term antitumor effect even when administered only once.

That is, the present invention includes the following (1) to (6).

(1) An expression cassette comprising the following (i), (ii) and (iii):
(I) a promoter selected from the group consisting of a promoter of a cbaH gene derived from a Bifidobacterium bacterium, a promoter of an rplJ gene derived from a Bifidobacterium bacterium, and a promoter of a hup gene derived from a Bifidobacterium bacterium,
(Ii) DNA encoding a signal sequence selected from the group consisting of the signal sequence of the BL1181 gene product derived from Bifidobacterium and the signal sequence of the amyB gene product derived from Bifidobacterium,
(Iii) DNA encoding a polypeptide or a cloning site for inserting the DNA

In the above expression cassette, DNA encoding a polypeptide is preferably inserted in (iii).

The promoter is preferably a promoter of cbaH gene derived from Bifidobacterium or hup gene product derived from Bifidobacterium. The signal sequence is preferably the signal sequence of the BL1181 gene product from Bifidobacterium.

The expression cassette comprises a BL1181 gene product derived from a bacterium belonging to the genus Bifidobacterium between (ii) a DNA encoding a signal sequence and (iii) a DNA encoding a polypeptide or a cloning site for inserting the DNA. A DNA encoding 1 to 20 consecutive amino acids following the signal sequence may be linked.

Alternatively, the expression cassette encodes the amino acid sequence shown in SEQ ID NO: 9 between (ii) a DNA encoding a signal sequence and (iii) a DNA encoding a polypeptide or a cloning site for inserting the DNA. DNA to be linked may be linked.

(2) An expression cassette comprising the following (i), (ii), (iii) and (iv):
(I) a promoter of a gene derived from a genus Bifidobacterium,
(Ii) DNA encoding a signal sequence of a gene product derived from a genus Bifidobacterium,
(Iii) DNA encoding 1 to 20 consecutive amino acids following the signal sequence of a gene product derived from a genus Bifidobacterium,
(Iv) DNA encoding a polypeptide or a cloning site for inserting the DNA

In the above expression cassette, it is preferable that DNA encoding the polypeptide is inserted in (iv).

The promoter is preferably selected from the group consisting of a promoter of cbaH gene derived from Bifidobacterium, rplJ gene derived from Bifidobacterium, and hup gene promoter derived from Bifidobacterium Is done.

In the expression cassette of (1) or (2) above, the polypeptide is preferably interleukin-10 (IL-10), interleukin-2 (IL-2), TRAIL / Apo2-L, type I interferon (IFN -β, etc.), interleukin-4 (IL-4) antagonistic mutants, and R-spondins.

The above expression cassette (1) or (2) includes, for example, a cbaH gene promoter derived from Bifidobacterium longum, DNA encoding the signal sequence of the BL1181 gene product derived from Bifidobacterium longum, IL- And DNA encoding 10.

The expression cassette of (1) or (2) above includes, for example, the cbaH gene promoter derived from Bifidobacterium longum or the hup gene promoter derived from Bifidobacterium longum, and the BL1181 gene derived from Bifidobacterium longum DNA encoding the product signal sequence, DNA encoding 1-20 contiguous amino acids following the signal sequence of the BL1181 gene product from Bifidobacterium longum, or DNA encoding LEISSTCDA, and IL-10 Containing the encoding DNA.

The expression cassette of (1) or (2) above includes, for example, the hup gene promoter derived from Bifidobacterium breve or the cbaH gene promoter derived from Bifidobacterium longum and the BL1181 gene derived from Bifidobacterium longum It contains DNA encoding the product signal sequence and DNA encoding IL-2.

The expression cassette of (1) or (2) above includes, for example, the hup gene promoter derived from Bifidobacterium breve or the cbaH gene promoter derived from Bifidobacterium longum and the BL1181 gene derived from Bifidobacterium longum Contains DNA encoding the product signal sequence, DNA encoding 1-20 contiguous amino acids following the signal sequence of the BL1181 gene product from Bifidobacterium longum, and DNA encoding IL-2 To do.

The above expression cassette (1) or (2) includes, for example, an rplJ gene promoter derived from Bifidobacterium longum, DNA encoding the signal sequence of the BL1181 gene product derived from Bifidobacterium longum, IL- 4 DNA encoding an antagonistic variant.

The expression cassette of the above (1) or (2) is, for example, a hup gene promoter derived from Bifidobacterium breve or a hup gene promoter derived from Bifidobacterium longum, and derived from Bifidobacterium addressensetis It contains DNA encoding the signal sequence of the amyB gene product and DNA encoding TRAIL / Apo2-L.

The expression cassette of (1) or (2) is, for example, a hup gene promoter derived from Bifidobacterium longum or a cbaH gene promoter derived from Bifidobacterium longum, and a BL1181 gene derived from Bifidobacterium longum It contains DNA encoding the product signal sequence and DNA encoding type I interferon.

The expression cassette of (1) or (2) is, for example, a hup gene promoter derived from Bifidobacterium longum or a cbaH gene promoter derived from Bifidobacterium longum, and a BL1181 gene derived from Bifidobacterium longum DNA encoding the product signal sequence, DNA encoding 1-20 contiguous amino acids following the signal sequence of the BL1181 gene product from Bifidobacterium longum or DNA encoding LEISSTCDA, and type I interferon Containing the encoding DNA.

The expression cassette of (1) or (2) above includes, for example, a cbaH gene promoter derived from Bifidobacterium longum, DNA encoding the signal sequence of the BL1181 gene product derived from Bifidobacterium longum, R- and DNA encoding spondins.

The expression cassette of (1) or (2) includes, for example, a cbaH gene promoter derived from Bifidobacterium longum, DNA encoding the signal sequence of the BL1181 gene product derived from Bifidobacterium longum, and bifido It contains DNA encoding 1-20 contiguous amino acids or DNA encoding LEISSTCDA following the signal sequence of BL1181 gene product derived from Bacterium longum, and DNA encoding R-spondins.

The expression cassette (1) or (2) is used for secreting and expressing a polypeptide in Bifidobacterium.

(3) An expression vector containing the expression cassette of (1) or (2) above.

(4) A recombinant microorganism comprising the expression cassette of (1) or (2) or the expression vector of (3).

The microorganism is, for example, a Bifidobacterium bacterium.

(5) A pharmaceutical composition comprising the microorganism of (4) above.

The above pharmaceutical composition can be used to treat a disease or disorder. Such disease or disorder is preferably inflammatory bowel disease or tumor disease.

(6) Production of a polypeptide comprising culturing a recombinant microorganism containing the expression cassette of (1) or (2) or the expression vector of (3), and collecting the polypeptide from the resulting culture Method.

According to the present invention, various useful polypeptides are efficiently secreted and expressed in an active state in Bifidobacterium having excellent characteristics in various points such as safety, pharmacokinetics and biological containment. A resulting expression cassette is provided. In addition, a microorganism that contains the expression cassette and secretes and expresses a useful polypeptide is provided. The microorganism can be used in a pharmaceutical composition for efficiently delivering a useful polypeptide to mucosa and / or tumor tissue by being administered to animals including humans. Thereby, treatment and / or prevention of a disease can be performed efficiently.

The map of pKKT427 is shown. The map of pBSM4 (A) and pBSM11 (B) is shown. An example of a structure of an expression cassette is shown. The western blot image of IL-10 in a culture supernatant is shown. Figure 2 shows in vitro biological activity of IL-10 in culture supernatant in MC / 9 cell proliferation assay. A graph is the activity of the positive control commercial IL-10 protein. A Western blot image (B. breve) of IL-10 in the culture supernatant is shown. The western blot image (pBSM4) of IL-10 in the culture supernatant is shown. The western blot image of IL-10 in the culture supernatant when the signal sequence and the insertion sequence after various lengths are used is shown. Figure 2 shows in vitro biological activity of IL-10 in culture supernatant in MC / 9 cell proliferation assay. A graph is the activity of the positive control commercial IL-10 protein. The graph of B shows the activity when the length of the insertion sequence after the signal sequence is 0 to 3. The graph of C shows the activity when the length of the insertion sequence after the signal sequence is 6 to 16. The western blot image of IL-2 in a culture supernatant is shown. Figure 2 shows in vitro biological activity of IL-2 in culture supernatant in CTLL / 2 cell proliferation assay. A graph is the activity of the positive control commercial IL-2 protein. The western blot image of IL-4 antagonistic variant in a culture supernatant or cytoplasm is shown. The western blot image (time-dependent change) of TRAIL / Apo2-L in a culture supernatant is shown. The western blot image (expression cassette A to D) of TRAIL / Apo2-L in a culture supernatant is shown. The western blot image (expression cassette A and E) of TRAIL / Apo2-L in a culture supernatant is shown. FIG. 5 shows in vitro biological activity of TRAIL / Apo2-L in culture supernatant in COLO 205 cell apoptosis assay. A graph shows the activity of a positive control commercially available recombinant solubilized hTRAIL / Apo2-L. The western blot image (pBSM11) of TRAIL / Apo2-L in a culture supernatant is shown. The western blot image of IFN-β in the culture supernatant or cytoplasm is shown. Fig. 5 shows in vitro biological activity of IFN-β in the culture supernatant in a type I interferon activity assay. The graph of A is the activity of the positive control commercial recombinant hIFN-β1a. The western blot image of R-spondin 2 dC in the culture supernatant or cytoplasm is shown. The in-vivo tumor growth inhibitory effect (4 times administration, arrow) of TRAIL / Apo2-L expression bifidobacterium (hup promoter) is shown. The weight change in the test of Example 11 is shown. The in-vivo tumor growth inhibitory effect (single administration, arrow) of TRAIL / Apo2-L expression Bifidobacterium (cbaH promoter) is shown.

Hereinafter, the present invention will be described in detail. This application claims priority of Japanese Patent Application No. 2009-108839 filed on Apr. 28, 2009, and includes the contents described in the specification and / or drawings of the above patent application. .

The present invention relates to an expression cassette that encodes a combination of a promoter and a signal sequence, and optionally further sequences following the polypeptide and / or signal sequence, useful for expressing and secreting the polypeptide in microorganisms, and uses thereof. .

In the present specification, an “expression cassette” means that a DNA encoding the polypeptide and various regulatory elements such as a promoter controlling the expression of the polypeptide are expressed as a host microorganism or a host. A DNA sequence that is ligated in such a way that it can function in cells. As used herein, “operably linked” refers to an expression cassette so that the DNA encoding the polypeptide is expressed under the control of a promoter and optionally under the control of other regulatory elements. Or it means that each component contained in the expression vector is linked.

The expression cassette according to the present invention includes a promoter. The “promoter” as used herein means a DNA sequence having a function of causing RNA polymerase to start transcription from a specific transcription start point (+1). In addition to this region, a region necessary for protein other than RNA polymerase to associate to regulate expression may be included. In the present specification, there is a case where it is described as “promoter region”, and this indicates a region containing a promoter as used herein.

In one embodiment of the present invention, the promoter includes a promoter of a cbaH gene derived from a Bifidobacterium bacterium, a promoter of an rplJ gene derived from a Bifidobacterium bacterium, and a promoter of a hup gene derived from a Bifidobacterium bacterium. Selected from the group consisting of The promoter of the hup gene derived from Bifidobacterium is preferably the promoter of the hup gene derived from Bifidobacterium breve.

Examples of the promoter of the cbaH gene derived from Bifidobacterium include the promoter region (SEQ ID NO: 1) of the cbaH gene of B. longum 105-A. This sequence is the promoter of the cbaH gene that encodes the conjugated bile acid hydrolase or bile 酸 塩 salt hydrolase of locus tag BL0796 contained in Bifidobacterium longum NCC2705, complete geneme of Accession AE014295 100% match with the area. It is also included in Bifidobacterium longum NCC2705 related nucleotide sequence SEQ ID 1102 of patent application publication EP1227152-A1 of Accession Number ABQ81846. It also matches 100% with the promoter region of the cbaH gene encoding the locus-tag 胆 BLD_0536 conjugated bile acid hydrolase contained in Bifidobacterium longum DJO10A, complete genome of Accession Number CP000605. It also matches 100% with the promoter region of the Bifidobacterium longum SBT2928 bile salt hydrolase (bsh) gene of Accession Number AF148138.

Examples of the promoter of the rplJ gene derived from Bifidobacterium include the promoter region (SEQ ID NO: 2) of the rplJ gene of B. longum ATCC-15707. This sequence is 100% identical to the promoter region of the rplJ gene encoding the 50S ribosomal protein L10 of locus tag BL1549 contained in Bifidobacterium longum NCC2705, complete genome of Accession Number AE014295. In addition, it matches 100% with the promoter region of rplJ gene encoding ribosomal protein L10 of locus tag BLD_1667 contained in Bifidobacterium longum DJO10A and complete genome of Accession Number CP000605.

Examples of the promoter of the hup gene derived from Bifidobacterium include the promoter region (SEQ ID NO: 3) of the hup gene of B. breve ATCC15700. In some cases, the promoter region (SEQ ID NO: 13) of the hup gene of B. longum ATCC15707 disclosed in Patent Document 1 (Japanese Patent No. 3642755) can also be used.

The promoter may be able to exhibit a desired promoter activity even if part of the sequence exemplified above or a further sequence is added to the whole or part of the sequence exemplified above. Therefore, in the present invention, a promoter containing all or part of the above-exemplified sequences (for example, the DNA sequences represented by SEQ ID NOs: 1, 2 and 3) can be used as a promoter. That is, as long as it has promoter activity, it may be any DNA fragment of the above-exemplified sequences (for example, the DNA sequences represented by SEQ ID NOs: 1 to 3), or the above-exemplified sequences (for example, represented by SEQ ID NOs: 1 to 3). Any DNA fragment containing a part of the DNA sequence).

In addition, the promoter may contain a mutation in one or several bases as long as it has a desired promoter activity. For example, deletion or substitution or addition of one or several, preferably 1 to 10, more preferably 1 to 5 bases in the sequence exemplified above (for example, the base sequences shown in SEQ ID NOs: 1 to 3) A promoter consisting of a sequence having Based on the sequence exemplified above, such a promoter can be produced by a method known in the art, for example, site-directed mutagenesis, so that variants having different sequences can be produced while maintaining the promoter activity.

In addition, for example, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the sequence exemplified above (for example, the base sequences shown in SEQ ID NOs: 1 to 3) is also used as long as it has promoter activity. be able to. The above-mentioned “DNA that can hybridize under stringent conditions” is about 70% or more, preferably about 80% or more, more preferably about about 80% or more of the sequence exemplified above (for example, the nucleotide sequences shown in SEQ ID NOs: 1 to 3). Examples thereof include DNA comprising a base sequence having a homology of 90% or more, most preferably about 95% or more. Here, stringent conditions refer to conditions in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, the sodium concentration is 10 mM to 300 mM, preferably 20 to 200 mM, and the temperature Means conditions at 25 ° C. to 70 ° C., preferably 42 ° C. to 55 ° C.

Here, “promoter activity” has the ability and function to initiate transcription of RNA from the DNA in the host when the DNA is linked to the promoter (region) in an expressible state and introduced into the host. Indicates. Whether or not a certain sequence has promoter activity can be confirmed by methods known in the art. For example, a vector in which various reporter genes such as luciferase gene (LUC), chloramphenicol acetyltransferase gene (CAT), β-glucuronidase (GUS) gene (uidA) and the like are linked to the downstream region of the promoter is prepared. Can be confirmed by measuring the expression of the reporter gene. As for the promoter activity, for example, approximately the same level of promoter activity (for example, about 0.01 to 100 times the activity) is maintained under the same conditions as those under which the promoter consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 3 functions. preferable. The production of a promoter having such a desired activity is known to those skilled in the art.

The expression cassette according to the present invention contains DNA encoding a signal sequence. The “signal sequence” as used herein is a peptide sequence contained in a protein molecule and is a structure that directs secretion of a polypeptide biosynthesized in the cytoplasm. This is also called a signal peptide, secretion signal, localization signal, or transport (transition) signal.

In one embodiment of the present invention, the signal sequence is selected from the group consisting of the signal sequence of the BL1181 gene product derived from Bifidobacterium and the signal sequence of the amyB gene product derived from Bifidobacterium.

For example, the signal sequence of the BL1181 gene product derived from Bifidobacterium genus bacteria includes the signal sequence (SEQ ID NO: 4) of the BL1181 gene product of B. longum 105-A. This is 100% consistent with the hypothetical signal sequence of Bifidobacterium longum NCC2705 hypothetical protein BL1181 of Accession Number NP_696350. It also matches 100% with the estimated signal sequence of COG3942: Surface antigen of Bifidobacterium longum DJO10A of Accession Number ZP_00121020. An example of DNA encoding the signal sequence shown in SEQ ID NO: 4 is DNA having the base sequence shown in SEQ ID NO: 6.

Also, for example, the signal sequence of the amyB gene product derived from Bifidobacterium genus bacteria includes the signal sequence (SEQ ID NO: 5) of the amyB gene product of B. adolescentis INT-57 disclosed in Non-Patent Document 18. This is a putative signal sequence of α-amylase, which is an amyB gene product of Bifidobacterium adolescentis of Accession Number AAP69820. This is also part of the protein-secretion-related alpha-amylase protein of Bifidobacterium sp. Protein-secretion-related alpha-amylase protein of SEQ ID ID NO2 of KR2004087056-A of Accession Number ADX45166. It is the Bifidobacterium adolescentis amylase signal の peptide of SEQ ID NO 1 of KR2006014660-A of Accession ADX45166 AEN03241. It is also part of Bifidobacterium adolescentis Amylase from SEQ ID NO 4 of KR2006014660-A of Accession Number AEN03244. It is 97% identical to the predicted signal sequence of hypothetical protein BIFADO_01864 in Bifidobacterium adolescentis L2-32 in Accession Number ZP_02029407. An example of DNA encoding the signal sequence shown in SEQ ID NO: 5 is DNA having the base sequence shown in SEQ ID NO: 7.

The signal sequence of the present invention includes variants of the above-exemplified sequences (for example, the sequences shown in SEQ ID NOs: 4 to 5) as long as they have activity as a desired signal sequence. Such variants also include corresponding sequences of orthologs of other species of Bifidobacterium.

Here, the activity as a signal sequence means a function that contributes to the translocation of the polypeptide to the endoplasmic reticulum membrane and the permeation of the polypeptide, and the polypeptide having the signal sequence is recognized by the signal recognition particle and enters the lumen of the endoplasmic reticulum. And is secreted through the membrane. Whether or not a sequence has activity as a signal sequence is determined by a method known in the art, for example, secretion of a linked polypeptide into a culture supernatant, and ELISA or Western blotting against the polypeptide. It can be confirmed by detecting using an antibody, or when the polypeptide has enzyme activity, by measuring the enzyme activity.

In addition, if a DNA encoding a sequence following a specific signal sequence or a DNA encoding LEISSTCDA is inserted between the DNA encoding the signal sequence and the DNA encoding the polypeptide, the secretion efficiency of the polypeptide is remarkably increased. May improve. In the present specification, in particular, a sequence having an activity of promoting the secretion of such a polypeptide is referred to as a “post-signal sequence insertion sequence”. Examples of the insertion sequence after the signal sequence include DNA encoding 1 to 20 consecutive amino acids following the signal sequence of the gene product (protein) derived from the genus Bifidobacterium.

SEQ ID NO: 8 is a peptide sequence consisting of 16 amino acids following the signal sequence of the BL1181 gene product of B. longum 105-A. This sequence is 100% identical to the predicted signal sequence of the Bifidobacterium thelongum NCC2705 hypothetical protein BL1181 of Accession Number NP_696350 and the predicted signal sequence of COG3942: surface antigen of Bifidobacterium longum DJO10A of Accession Number ZP_00121020. An example of DNA encoding the signal sequence post-insertion sequence shown in SEQ ID NO: 8 is DNA having the base sequence shown in SEQ ID NO: 10.

In addition, a peptide sequence consisting of amino acids LEISSTCDA (SEQ ID NO: 9) (hereinafter referred to as LEISS) can be used as the insertion sequence after the signal sequence. This sequence is an artificial sequence shown in a paper on the secretion of heterologous proteins in L. Lactis (Le Loir Y, et al. J Bacteriol. 1998. 180 (7): 1895-903.). An example of DNA encoding the signal sequence post-insertion sequence shown in SEQ ID NO: 9 is DNA having the base sequence shown in SEQ ID NO: 11.

As the insertion sequence after the signal sequence of the present invention, DNA encoding any number of amino acids among 1 to 20 consecutive amino acids following the signal sequence can be used as long as it has a desired secretion promoting activity. For example, DNA encoding preferably 1 to 18, more preferably 1 to 16 consecutive amino acids can be mentioned. In addition, variants of the sequences exemplified above (for example, the sequences shown in SEQ ID NOs: 8 to 9) can also be used. Such mutants also include deletion mutants and extension mutants. Furthermore, the variants also include corresponding sequences of orthologs of other species of Bifidobacterium.

Whether or not the sequence or variant here can be used as the insertion sequence after the signal sequence in the present invention is determined by, for example, linking the candidate sequence to the signal sequence, followed by the polypeptide in the culture supernatant. The increase in secretion amount can be confirmed by detecting by ELISA or Western blotting using an antibody against the polypeptide, or when the polypeptide has enzyme activity, by measuring the enzyme activity.

The expression cassette according to the present invention contains DNA encoding a polypeptide or a cloning site for inserting such DNA for the expression and secretion of the polypeptide.

“Polypeptide” as used herein is not particularly limited as long as it is a polypeptide composed of two or more amino acids, and means a polypeptide having any function / activity derived from any organism.

Specific examples of such polypeptides include protein hormones or peptide hormones such as growth hormone, prolactin, luteinizing hormone, parathyroid hormone, thyroid stimulating hormone, insulin, somatostatin, calcitonin, vasoactive intestinal polypeptide Peptides, adrenomedullin, leptin, glucagon-like peptide (GLP) -1 ~ 2 or insulin-like growth factor (IGF) -1 ~ 2, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte / macrophage Colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), hepatocyte factor (SCF), thrombopoietin (TPO), leukocyte inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic Factor (CNTF), Interleukin (IL) 1-35, I-III type inter Geron (IFN), tumor necrosis factor (TNF) superfamily proteins such as TNF-α, CD40, TRAIL / Apo2-L, FAS ligand or RANKL, chemokines such as CCL1-28 or CXCL1-17 fibroblasts Growth factor (FGF) 1-23, hepatocyte growth factor (HGF), insulin-like growth factor (IGF) -1 -2, keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), transforming growth factor ( TGF) -β superfamily proteins such as TGF-β or bone morphogenetic proteins (BMP) 1-15, vascular endothelial growth factor (VEGF) families such as VEGF-A to -E, neurotrophic factors such as nerve growth Factors (NGF), such as R-spondin 1-4, Trefoil factor 1-3, epidermal growth factor (EGF) family proteins such as EGF or neuregulin-1-4, connective tissue growth factor (CTGF), tissue plasminogen activation Examples include, but are not limited to, factor (t-PA), urokinase, and immunoglobulins such as camelid VHH antibodies.

Alternatively, the “polypeptide” may be a receptor or antagonist for the above polypeptide.

Alternatively, a “polypeptide” is an intracellular protein or enzyme, such as a prodrug converting enzyme, such as E. coli or yeast cytosine deaminase (CD) or glucuronidase, and an antigenic polypeptide (ie, a subject whose immune response is elicited). ), And luminescent proteins (such as luciferase), and toxins such as Pseudomonas aeruginosa exotoxin (PE).

Furthermore, the “polypeptide” may be a partial peptide, a mutant, or a fusion of the above polypeptide. For example, it may be a fusion polypeptide of CD or p53 and a protein transduction domain (PTD) such as VP22 or TAT.

In one embodiment of the invention, the polypeptide is IL-10, IL-2, IL-4 antagonist mutant, TRAIL / Apo2-L, type I IFN (IFN-α, β, ω) or R-spondins. Preferably there is.

IL-10 is an anti-inflammatory cytokine. To date, clinical trials of recombinant IL-10 have been attempted in inflammatory bowel disease, but have not been very successful (World J Gastroenterol. 2004 Mar 1; 10 (5): 620-5.). On the other hand, L. lactis, which secretes IL-10, may have advantages such as IL-10 acting only in the gastrointestinal tract by oral administration, and clinical trials for inflammatory bowel disease have been conducted. (Non-Patent Document 2: Remaut E, et al. Bioscience and Microflora. 2006. 25 (3): 81-97.) Therefore, Bifidobacterium that secretes and expresses IL-10 may be a therapeutic drug for inflammatory bowel disease. L. lactis that persistence in the intestinal tract may be excellent (Non-patent document 9: Watson D, et al. BMC Microbiol. 2008. 8: 176.) And that it is an obligate anaerobe. Can be an advantage of Bifidobacterium. It may also be useful for diseases in other mucosal sites.

IL-2 can proliferate NK cells and T cells and activate antitumor immunity. Local administration of IL-2 has been effective in many clinical trials (Den Otter W, et al. Cancer Immunol Immunother. 2008. 57 (7): 931-50.). There is also an animal model report that IL-2 has no antitumor effect when administered systemically, but is effective when delivered locally to the tumor (MarlindlinJ, et al. Clin Cancer Res. 2008. 14 (20): 6515 -twenty four.). Therefore, Bifidobacterium survives and grows only locally when administered intravenously (Kimura 腫 瘍 NT, et al. Cancer Res. 1980. 40 (6): 2061-8.) And expresses IL-2 Bifidobacterium is considered to be an effective treatment for tumors. In addition, Bifidobacterium that secretes and expresses IL-2 may be used as an adjuvant for vaccines.

Many epithelial cancer cells and cancer stem cells are resistant to apoptosis induced by TRAIL / Apo2-L, which has been suggested to be mediated by tumor cell-derived IL-4 (TodaroToM, et al. Cell Death Differ. 2008. 15 (4): 762-72., Todaro M, et al. Cell Stem Cell. 2007. 1 (4): 389-402.). Thus, IL-4 antagonistic mutants may be useful for the treatment of tumors. In addition, since IL-4 is involved in many immune responses, IL-4 antagonistic mutants may be useful for immune diseases. Furthermore, IL-4 antagonistic mutants may be able to achieve lower toxicity with higher efficacy by allowing them to selectively act in tumors or mucosal areas by being secreted and expressed in Bifidobacterium.

TRAIL / Apo2-L interacts with receptors DR4 and / or DR5 on tumor cells and induces apoptosis in tumor cells (Ashkenazi A, et al. Nat Rev Drug Discov. 2008. 7 (12): 1001 -12.) Therefore, agonists of DR4 and DR5 including TRAIL / Apo2-L are considered useful for tumor treatment, and clinical trials are being conducted. Bifidobacterium that secretes and expresses TRAIL / Apo2-L may have an advantage as a therapeutic agent in terms of side effects and persistence by selectively targeting to a tumor.

Type I IFN exerts its antitumor effect on tumors either directly or through indirect action through angiogenesis inhibition or immunity (Gresser I. Biochimie. 2007. 89 (6-7): 723-8., Ferrantini M Et al. Biochimie. 2007. 89 (6-7): 884-93.). In addition, type I IFN is widely used as an antiviral agent. From these facts, Bifidobacterium that secretes and expresses type I IFN can also be used as an antitumor agent and an antiviral agent.

Administration of R-spondin 1 to 4 (R-spondins) has similar biological activity that leads to intestinal hypertrophy (Kim KA, et al. Cell Cycle. 2006. 5 (1): 23-6.) R-spondin 1 is effective in an inflammatory bowel disease model (Zhao J, et al. Gastroenterology. 2007. 132 (4): 1331-43.). This suggests that R-spondins may be used to treat intestinal diseases. As with IL-10, Bifidobacterium that secretes and expresses R-spondins may have advantages over R-spondin protein formulations. Furthermore, it may be used to treat diseases of mucosal regions other than the intestines (ZhaoZJ, et al. Proc Natl Acad Sci U S A. 2009. 106 (7): 2331-6.).

When targeting humans in the treatment or prevention of diseases or disorders as described below, IL-10, IL-2, IL-4 antagonistic mutants, TRAIL / Apo2-L, type I IFN (IFN-α, β, ω) or R-spondins are preferably human orthologs.

全 て All the above-mentioned polypeptides are known, and those skilled in the art can appropriately obtain their amino acid sequences and base sequences encoding them from known literature or public databases (GenBank, etc.).

Alternatively, the expression cassette according to the present invention may contain a cloning site for inserting a DNA encoding a polypeptide. Such cloning sites are known in the art, and for example, multiple cloning sites containing sites recognized by various restriction enzymes can be utilized. In the case of an expression cassette containing such a cloning site, it becomes easy to insert DNA encoding a desired polypeptide into the expression cassette via the cloning site.

The combination of the above components can be selected so as to achieve desired secretory expression depending on the type of polypeptide to be expressed, the type of vector used, the type of host microorganism, and the like. For example, a DNA encoding an appropriate promoter and signal sequence is ligated with DNA encoding a polypeptide to be expressed, an expression vector is prepared, the expression vector is introduced into an appropriate microorganism, and Whether the desired secretory expression is achieved can be confirmed by measuring the expression level of the peptide, preferably the secretion level.

Examples of, but not limited to, combinations of promoter and signal sequences that can achieve optimal secretory expression, and optionally post-signal sequence insertion sequences, for a particular polypeptide:
(I) For IL-10 expression, the following is preferred:
A combination of the cbaH gene promoter from Bifidobacterium longum and the signal sequence of the BL1181 gene product from Bifidobacterium longum, or the cbaH gene promoter or Bifidobacterium longum from Bifidobacterium longum A combination of the hup gene promoter derived from the signal sequence of the BL1181 gene product derived from Bifidobacterium longum and the signal sequence after insertion of the BL1181 gene product derived from Bifidobacterium longum or a sequence encoding LEISSTCDA.

(Ii) For IL-2 expression, the following is preferred:
Combination of hup gene promoter from Bifidobacterium breve or cbaH gene promoter from Bifidobacterium longum and signal sequence of BL1181 gene product from Bifidobacterium longum, or Bifidobacterium breve Hup gene promoter derived from or cbaH gene promoter derived from Bifidobacterium longum, signal sequence of BL1181 gene product derived from Bifidobacterium longum, and signal sequence of BL1181 gene product derived from Bifidobacterium longum A combination of insert sequences.

(Iii) For the expression of an IL-4 antagonistic mutant, a combination of the rplJ gene promoter derived from Bifidobacterium longum and the signal sequence of the BL1181 gene product derived from Bifidobacterium longum is preferred.

(Iv) The expression of TRAIL / Apo2-L includes the hup gene promoter derived from Bifidobacterium breve or the hup gene promoter derived from Bifidobacterium longum, and the amyB gene derived from Bifidobacterium adrecentis A combination with the product signal sequence is preferred.

(V) For expression of type I interferons, particularly IFN-β, the following is preferred:
Combination of hup gene promoter from Bifidobacterium longum or cbaH gene promoter from Bifidobacterium longum and signal sequence of BL1181 gene product from Bifidobacterium longum, or Bifidobacterium longum Hup gene promoter derived from or cbaH gene promoter derived from Bifidobacterium longum, signal sequence of BL1181 gene product derived from Bifidobacterium longum, and signal sequence of BL1181 gene product derived from Bifidobacterium longum Insertion sequence or combination of sequences encoding LEISSTCDA.

(Vi) For expression of R-spondins, particularly dC type R-spondin2, the following is preferred:
A combination of the cbaH gene promoter from Bifidobacterium longum and the signal sequence of the BL1181 gene product from Bifidobacterium longum, or the cbaH gene promoter from Bifidobacterium longum, and Bifidobacterium A combination of a signal sequence of the BL1181 gene product derived from longum and a signal sequence insert after the signal sequence of the BL1181 gene product derived from Bifidobacterium longum or a sequence encoding LEISSTCDA.

発 現 In addition to the above-described components, the expression cassette according to the present invention may contain a cis element such as an enhancer sequence, a selection marker, a ribosome binding sequence (SD sequence), a homologous sequence, and the like as desired.

A “terminator sequence” is a DNA sequence having an activity to terminate transcription, and is preferably linked downstream of DNA encoding a polypeptide, but is not necessarily required.

In one embodiment of the present invention, the terminator sequence of the hup gene of B. longum ATCC -15707 (Tanaka K, et al. Biosci Biotechnol Biochem. 2005. 69 (2): 422-5.) Is used as the terminator sequence. Although other terminator sequences known in the art can be used.

“Selection marker” is used to select only transformed cells. Examples include drug resistance markers such as ampicillin resistance, chloramphenicol resistance, erythromycin resistance, neomycin resistance, spectinomycin resistance, or tetracycline resistance. Preferably, a spectinomycin resistance gene (SpR) or a chloramphenicol resistance gene (CmR or cat) is used. In order to select only transformed cells, it is not always necessary to use a drug resistance marker. For example, detection by auxotrophy, detection of an introduced expression cassette or expression vector by PCR, or polypeptide Transformed cells can also be selected by detecting the product by ELISA or Western blotting.

In addition, when the polypeptide is presented on the surface of a microbial cell, it is preferable to connect the surface presentation sequences. As the surface layer display sequence, for example, a transmembrane anchor sequence of PgsA, which is a protein involved in the biosynthesis of poly-γ-glutamic acid of Bacillus issubtilis (Narita J, et al. Appl Environ Microbiol. 2006. 72 (1): 269-75.) And LPXTG cell wall anchoring motif and peptidoglycan binding domain (Ramasamy R, et al. Vaccine. 2006. 24 (18): 3900-8.) Are also known.

Methods for linking the above-described constituent elements are known in the art. For example, a method in which purified DNA of each constituent element is cleaved with an appropriate restriction enzyme and ligated or the like is used. Can do. Alternatively, a method of linking by a synthetic PCR method can be employed.

The present invention also provides an expression vector containing the above expression cassette. As used herein, “expression vector” refers to a vector into which a promoter for transcription of DNA encoding a target polypeptide and an expression unit (expression cassette) composed of other appropriate sequences are inserted. In which DNA encoding the polypeptide is inserted. In the present specification, the term “vector” refers to a nucleic acid molecule used for gene recombination technology for introducing a nucleic acid molecule inserted therein into a host cell, and the vector is not limited thereto. Examples include plasmid vectors, phage vectors, phagemid vectors, and artificial chromosomes.

In one embodiment of the invention, the vector is pTB6 (Matsumura H, et al. Biosci Biotechnol Biochem. 1997. 61 (7): 1211-2., Tanaka K, et al. Biosci Biotechnol Biochem. 2005. 69 (2 ): 422-5.) The shuttle vector pKKT427 of Bifidobacterium containing the replicon and Escherichia coli (Fig. 1; Yasui K, et al. Nucleic Acids Res. 2009. 37 (1): e3.) There is no particular limitation.

In another embodiment of the present invention, the vector is pTB4 (Imamoto F and Kano Y, “Bifizusukin no Kenkyu” (in Japanese), ed. By Mitsuoka T, JapanBifidus Foundation, Tokyo, Japan, 1994, pp. 150 157.) replicon-containing Bifidobacterium and E. coli shuttle vectors pBSM4 and pBSM11 (FIGS. 2A and B). Here, pBSM4 and pBSM11 are obtained by reversing the directions of the multiple cloning sites.

To insert each component or expression cassette into a vector, for example, the purified DNA is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site of a vector DNA or a multicloning site, and ligated to the vector. It is necessary to construct the vector such that the vector is autonomously replicated in the host microorganism (extrachromosomal) or that the DNA on the vector is integrated into the genome of the host microorganism (intrachromosome).

In the expression vector, the expression cassette to be linked or each component may be singular or plural. Moreover, the expression vector introduced into the host microorganism may be one type or plural types.

For basic genetic engineering procedures for the construction of expression vectors (nucleic acid constructs), commercially available experiments such as Molecular 、 Cloning, 2nd ed. (Sambrook, J et al. 1989. Cold Spring Harbor Laboratory Press.) Or Molecular Cloning , 3rd ed. (Sambrook, Jet al. 2001. Cold Spring Laboratory Press.) And the like.

Furthermore, the present invention provides a recombinant microorganism comprising the above expression cassette or the above expression vector. The “recombinant microorganism” referred to in the present specification is a microorganism that includes the expression cassette or expression vector (nucleic acid construct) of the present invention and is capable of secreting and expressing the polypeptide encoded by the expression cassette or expression vector. The host microorganism that can be used for the production of such a recombinant microorganism is not particularly limited as long as it is a microorganism that can secrete and express the polypeptide from the introduced expression cassette or expression vector. It is a bacterium. Gram-positive bacteria include, for example, Bifidobacterium bacteria, Lactococcus bacteria, Lactobacillus bacteria, Clostridia bacteria, Bacillus bacteria, and the like. More preferably, the microorganism is a Bifidobacterium bacterium. Alternatively, the microorganism includes Gram-negative bacteria such as Salmonella (Enterobacteriaceae), Escherichia.

The Bifidobacterium bacterium used in the present invention may be any strain belonging to the genus. For example, B. adolescentis, B. animalis, B. bifidum, B. breve ), Bifidobacterium catenulatum (B. catenulatum), B. フ ィ dentium, B.infantis, Bifidobacterium lactis (B. イ ン lactis) ), Bifidobacterium longum (B. longum), Bifidobacterium pseudolongum (B. pseudolongum), Bifidobacterium thermophilum (B. thermophirum) and the like. All of these bacteria are commercially available or can be easily obtained from depository institutions. Alternatively, these bacteria can be collected from a living body. Furthermore, these mutants or bacteria bred from them may be used.

In one embodiment of the present invention, the Bifidobacterium is B. longum 105-A (Matsumura H, et al. Biosci Biotechnol Biochem. 1997. 61 (7): 1211-2.) Or B.breve ATCC. 15700 (available from American Type Culture Collection (ATCC)).

For culture of recombinant microorganisms, any of natural and synthetic media can be used as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. and can efficiently culture transformants. Well, those skilled in the art can appropriately select a known medium suitable for the strain to be used. For culturing Bifidobacterium, for example, a sterilized medium having the following composition can be used.

"MRS liquid medium"
Lactobacilli MRS Broth (Difco Laboratories, Detroit, MI)
50 mM Sucrose
3.4 mg / ml L-Ascorbic Acid Sodium Salt
0.2 mg / ml L-Cysteine Hydrochloride
The “MRS agar medium” is a mixture of Bacto Agar (Difco Laboratories) in the MRS liquid medium at a ratio of 1.5%. “MRS-HEPES medium” is a 1: 1 mixture of the above MRS liquid medium and 0.5 M HEPES Buffer Solution (pH 7.2 to 7.5). The transformed bacteria having a drug resistance marker can be selectively cultured by adding various drugs according to the selection marker. For example, spectinomycin is used at 100 μg / ml and chloramphenicol is used at 2.5 μg / ml.

Bifidobacterium is cultured under anaerobic conditions at about 30 to 40 ° C., preferably about 37 ° C. Anaerobic conditions mean a hypoxic environment in which Bifidobacterium can grow. For example, an oxygen scavenger such as an anaerobic chamber, an anaerobic box or an anero pack (Mitsubishi Gas Chemical, Tokyo) is added. It can be obtained by using a sealed container or a bag.

Any known method can be used as a transformation method for introducing an expression cassette or expression vector into a host microorganism. For example, an electroporation method, a protoplast method, etc. are mentioned. In the present invention, it is preferable to use an electroporation method. The transformant can be isolated as a colony using, for example, a medium containing a drug corresponding to the selection marker (for example, MRS agar medium in the case of Bifidobacterium).

The recombinant microorganism containing the expression cassette or expression vector according to the present invention produced as described above expresses and secretes the polypeptide encoded by the expression cassette or expression vector at a high level. As used herein, “secretion” means that a cell excretes a metabolite out of the cell, but also includes presentation to the cell surface. Preferably, the polypeptide is secreted and expressed on the order of nanogram (ng) to microgram (μg) per ml. Therefore, an expression vector containing the expression cassette according to the present invention and / or a recombinant microorganism containing the expression vector can be used to deliver the polypeptide. For example, since Bifidobacterium is known to localize to mucosa and / or tumor tissue or cells when administered to humans, recombinants comprising the expression cassette or expression vector according to the present invention Bifidobacterium is useful for local delivery of the polypeptide encoded thereby to mucosa and / or tumor tissue or cells.

Therefore, the present invention also provides a pharmaceutical composition comprising the recombinant microorganism according to the present invention. The pharmaceutical composition according to the present invention is a solution or suspension, or a granular or powdery dried product as it is, more generally, a recombinant microorganism as an active ingredient and one or more pharmaceutical additives. In the form of a composition comprising

The pharmaceutical composition according to the present invention can be used, for example, for gene therapy using symbiotic bacteria as a host.

In the pharmaceutical composition according to the present invention, the recombinant microorganism can be used in a live state or a non-live state.

The recombinant microorganism may be subjected to a known post-treatment. For example, if desired, after crude purification by centrifugation or the like, it may be dissolved or suspended in a suitable solvent. Further, if desired, freeze-drying or spray-drying may be performed to form a powdery product or a granular product.

The pharmaceutical composition according to the present invention can be made into an appropriate dosage form according to a desired administration route. Examples of preparations suitable for oral administration include, for example, tablets, granules, fine granules, powders, syrups, solutions, capsules or suspensions. Examples of preparations suitable for parenteral administration include injections, drops, inhalants, sprays, suppositories, transmucosal absorbents, and the like. Such a composition can itself be produced according to a method well known or commonly used in the field of pharmaceutics.

The pharmaceutical composition according to the present invention may be blended in, for example, food or feed. The form and type of food with which the pharmaceutical composition is blended are not particularly limited. For example, it can be blended in various forms of food such as solid food, jelly food, liquid food, capsule food, dairy products (yogurt, etc.).

The pharmaceutical composition according to the present invention can be administered to a living body for the purpose of treating or preventing various diseases and disorders. The subject to be administered with the pharmaceutical composition according to the present invention is not limited to humans, and vertebrates, specifically mammals such as primates (monkeys, chimpanzees, etc.), domestic animals (bovines, horses, Pigs, sheep, etc.), pet animals (dogs, cats, etc.), laboratory animals (mouse, rats, etc.), and also reptiles and birds.

The pharmaceutical composition according to the present invention is administered for the treatment or prevention of digestive tract diseases such as digestive tract diseases such as the small intestine (duodenum, jejunum, ileum), and large intestine (cecum, colon, rectum). More preferably, inflammatory bowel disease, such as peptic ulcer (duodenal ulcer), gastrointestinal mucositis, ulcerative colitis, non-infectious colitis, infectious colitis, Crohn's disease, preferably ulcerative colitis And applied to Crohn's disease.

The pharmaceutical composition according to the present invention is administered for the treatment or prevention of tumors, more preferably various solid cancers. Examples of solid cancer include colon cancer, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, islet cell cancer, choriocarcinoma, colon cancer, renal cell cancer, adrenal cortex Cancer, bladder cancer, testicular cancer, prostate cancer, testicular tumor, ovarian cancer, uterine cancer, choriocarcinoma, thyroid cancer, malignant carcinoid tumor, skin cancer, malignant melanoma, osteosarcoma, soft tissue sarcoma, neuroblastoma, Wilms Examples include tumors, retinoblastoma, melanoma, and squamous cell carcinoma.

疾患 The disease or disorder to be treated or prevented by the pharmaceutical composition according to the present invention may be a single disease, a concurrent disease, or a disease other than the above.

Also, the pharmaceutical composition according to the present invention can be used as a vaccine or an adjuvant for immunity induction accompanying the vaccine.

The administration route of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include oral administration and parenteral administration. Parenteral administration can include administration routes such as intratracheal, rectal, subcutaneous, intramuscular, intravenous, intraarterial and affected area, for example, intratumoral.

The dosage and administration frequency of the pharmaceutical composition of the present invention are not particularly limited, and depend on various conditions such as the type of pathological condition to be treated, the administration route, the age and weight of the patient, the symptoms, and the severity of the disease. It is possible to select appropriately.

The pharmaceutical composition of the present invention may be used in combination with other medicines, treatment or prevention methods. Such other medicaments may form one preparation with the pharmaceutical composition of the present invention, or may be administered in separate preparations at the same time or at intervals.

Although the pharmaceutical composition according to the present invention cannot be metabolized by the animal itself, it may be used in combination with a nutrient source that can be used by the recombinant microorganism contained in the pharmaceutical composition. For example, lactulose or lacto N biose can be used as a growth promoter for Bifidobacterium.

組 換 え Recombinant microorganisms contained in the pharmaceutical composition according to the present invention can be killed with an appropriate antibiotic. Accordingly, after the pharmaceutical composition according to the present invention is administered to a subject, the recombinant microorganism can be removed from the subject body by administering an antibiotic to the subject after a certain period of time.

Of course, the expression cassette, the expression vector and the recombinant microorganism according to the present invention can of course be used for industrial production of a desired polypeptide. Specifically, the polypeptide can be produced by culturing a recombinant microorganism containing the expression vector according to the present invention and collecting the polypeptide from the resulting culture. When the polypeptide is secreted outside the cells after culturing, the culture solution is used as it is, or the cells are removed by centrifugation or the like. When the polypeptide is produced in the cells or on the cell surface of the cells, the protein is extracted by crushing the cells. Polypeptides can be isolated by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination. Can be purified.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples. From the results of the following examples, the secretory expression of heterologous polypeptides, particularly the secretory expression of polypeptides with biological activity, is a component of the expression cassette, i.e., the promoter region and signal sequence, and optionally the insertion sequence after the signal sequence It can be seen that each is greatly affected.

Recombinant B. longum 105-A secreting mouse IL-10
(1) Construction of mouse IL-10 secretion expression vector HindIII of the multicloning site (MCS) of the pTB6-based shuttle vector pKKT427 (provided by Professor Yasumasa Kano, Kyoto Pharmaceutical University; Fig. 1) An expression cassette was inserted between and NotI. The expression cassette is ligated in the order of HindIII, promoter region, signal sequence, post-signal sequence insertion sequence, BamHI site, mouse IL-10 cDNA, stop codon, SpeI site, terminator, and NotI site (FIG. 3). Therefore, the amino acid GS encoded by BamHI's GGATCC is inserted into the expressed protein. In addition, mouse IL-10 cDNA is obtained by removing the original signal sequence. Here, referring to the literature (Ball C, et al. Eur Cytokine Netw. 2001. 12 (1): 187-93.) That it is better to remove the unwanted reaction of cysteine unnecessary for activity, the 149th A mutant in which cysteine was replaced with tyrosine (mIL-10 C149Y) was used (SEQ ID NO: 15). As the terminator sequence, SEQ ID NO: 12 was used in which a single base mutation was added to the SpeI site originally possessed by the sequence based on the terminator of the hup gene of B. longum ATCC 15707. As a comparative control, the promoter region (SEQ ID NO: 13) of the hup gene of B. longum ATCC15707, which is the promoter region of Patent Document 1 (Patent No. 3642755), was used. Table 1 shows combinations of expression cassettes up to the BamHI site.

The procedure for constructing the expression cassette is as follows. A terminator sequence with SpeI and NotI sites added at the ends was artificially synthesized by PCR. The synthetic DNA fragment was digested with restriction enzymes SpeI and NotI and inserted between SpeI and NotI at the multicloning site of pBluescript II (Stratagene, LaLa Jolla, CA). MIL-10 C149Y cDNA with BamHI site and SpeI site added at the ends was synthesized by introducing mutations by PCR using mouse IL-10 cDNA (Open BioSystems, Huntsville, AL) as a template. The PCR-amplified DNA fragment was digested with restriction enzymes BamHI and SpeI, and inserted between BamHI and SpeI of the multiple cloning site of pBluescript II where the terminator had already been inserted. Then, a fragment from BamHI to NotI containing cDNA and terminator was transferred between BamHI and NotI of the multicloning site of pKKT427 from pBluescript II. The promoter region was amplified by PCR from the B. longumum genome. The signal sequence of BL1181 and the insertion sequence after the signal sequence were also amplified from the B. longumum genome by PCR. The signal sequence of amyB was artificially synthesized by the PCR method. From the promoter region to the signal sequence, or from the signal sequence to the insertion sequence, the PCR amplified DNA fragment was used as a template, a HindIII site and a BamHI site were added to the ends, and synthesis was performed by PCR. The PCR amplified DNA fragment was digested with restriction enzymes HindIII and BamHI, and inserted between HindIII and BamHI of the multicloning site of pKKT427 in which cDNA and terminator had already been inserted. Then, a mouse IL-10 secretion expression vector was completed. As the PCR enzyme, KOD Plus Ver. 2 (Toyobo, Osaka, Japan) was used. All the expression cassettes of the completed vector were confirmed in sequence.

(2) Detection of mouse IL-10 protein in culture supernatant of recombinant B. longum 105-A pKKT427 containing each of the expression cassettes A to H in Table 1 was isolated from B. longum 105-A (former Kyoto Pharmaceutical University Transformed by Professor Yasumasa Kano). Electroporation for transformation of Bifidobacterium was performed under conditions of 12 kV / cm, 25 μF, and 200 Ω. Moreover, although the MRS agar medium was used as a culture medium for culturing Bifidobacterium, this is an MRS liquid medium (Lactobacilli MRS Broth (Difco Laboratories, Detroit, MI), 50 mM sucrose, 3.4 mg / ml. L-ascorbic acid sodium salt and 0.2 mg / ml L-cysteine hydrochloride) are mixed so that Bacto Agar (Difco Laboratories) has a ratio of 1.5%. When spectinomycin was added, it was added at 100 μg / ml.

Four colonies were picked for each expression cassette from colonies that had grown on the spectinomycin-containing MRS agar medium. Each clone is pre-cultured in spectinomycin-containing MRS liquid medium, diluted with MRS-HEPES medium (MRS liquid medium and 0.5 M HEPES Buffer Solution (pH 7.2-7.5) mixed 1: 1), 600nm Was diluted to 0.1 with an absorbance of OD (OD600 nm). After culturing at 37 ° C. for 6 hours, the culture supernatant and cells were collected. The cells were washed once with PBS, suspended in the same amount of PBS as the culture supernatant, and sonicated.

10 μl each of the culture supernatant and cell disruption supernatant was subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, transferred to a PVDF membrane, and mouse IL-10 was detected by Western blotting. As a primary antibody, an anti-mouse IL-10 antibody (PeproTech, Rocky Hill, NJ) was used, and an ECL plus western blotting detection reagent (GE Healthcare UK UK Ltd., Little Chalfont, Buckinghamshire, England) was used. As a negative control (Ctrl), the culture supernatant of B. longum 105-A transformed with pKKT427 without an expression cassette was used. Commercially available recombinant mIL-10 組 換 え C149Y (R & D （Systems, Minneapolis, MN) was used as a positive control.

FIG. 4 shows a Western blot image of IL-10 in the culture supernatant obtained with an image analyzer LAS-3000 (Fujifilm, Tokyo, Japan). As clearly shown here, a band was detected at the expected molecular weight, and the order of expression was A <H <B <E <C <D <F <G. There was almost no difference between 4 clones of the same expression cassette (data not shown). In particular, clones (C, D, F, G) using the insertion sequence after the signal sequence contained more IL-10 protein in the supernatant than in the cells (data not shown).

Each band on the Western blot image was analyzed with the software attached to LAS-3000, Image Gauge Ver. 4.0 (Fujifilm, Tokyo) using the commercially available recombinant mouse IL-10 band as a positive control as a standard. As shown in Fig. 1, the IL-10 concentration in the culture supernatant was calculated. As a conventional technique, secretory expression of human IL-10 using the promoter of B. longum NCC 2705 hup gene and the signal sequence of B. bifidum DSM 20215 β-galactosidase has been reported. The detected concentration was 22 pg / ml (Non-patent Document 15: Reyes Escogido ML, et al. Biotechnol Lett. 2007. 29 (8): 1249-53.). Compared to this, it is clear that this system is far superior. Further, the effects of the combination of the promoter and the signal sequence and the presence or absence of the insertion sequence after the signal sequence are clearly shown.

(3) Bioactivity measurement of mouse IL-10 in culture supernatant of recombinant B. longum 105-A Mouse IL-10 in culture supernatant was determined by proliferation assay using mouse mast cell line MC / 9 (ATCC CRL-8306). A biological activity of -10 was measured. First, the culture supernatant was buffer exchanged with DMEM medium by gel filtration using a NAP column (GE Healthcare). MC / 9 cells were seeded at 1 × 10 ⁴ cells / well in a 96-well plate. A buffer exchange solution or a dilution obtained by diluting the buffer exchange solution with the same buffer was added to each well in a volume of 10% of the MC / 9 cell culture solution. The final concentration of the buffer exchanged solution in the MC / 9 cell culture solution was adjusted to 0 to 10% (v / v). Cultured for 72 hours and assayed using CellTiter-Glo (Promega, Madison, Wis.). As a negative control (Ctrl), a culture supernatant of B. longum 105-A transformed with pKKT427 without an expression cassette was used. Commercially available recombinant mIL-10 C149Y (R & D Systems) was used as a positive control.

The results of the soot assay were as shown in FIG. The addition of 10% of the buffer exchanged solution is thought to be due to the presence of many components in the culture supernatant, but the growth of the growth promoting effect was suppressed. When calculated with the addition of 2%, the specific activities of F and G were 196% and 42%, respectively, for the commercially available mIL-10 C149Y protein (FIG. 5A) (FIG. 5B). When calculated with the value of 10% addition, E was 127% with respect to F. Therefore, E without using the signal sequence post-insertion sequence and F using BL1181 as the signal sequence post-insert sequence were inferior in biological activity to commercially available mIL-10 C149Y. On the other hand, G using LEISS as the insertion sequence after the signal sequence had low biological activity.

From the above results, it can be seen that the present invention makes it possible to secrete and express IL-10 in Bifidobacterium at a level far exceeding that of the prior art. The cbaH promoter region was superior to the B. longum hup promoter region, where IL-10 protein secretion expression and other heterologous protein intracellular expression were reported. In addition, the signal sequence of BL1181 was superior to the signal sequence of amyB, which has been reported to be used for secretory expression of other heterologous proteins. In addition, the secretion promoting effect of the insertion sequence after the signal sequence was both remarkable. However, in terms of biological activity of IL-10, it was better to use the insertion sequence after the signal sequence of BL1181.

B. breve ATCC 15700 secretes mouse IL-10
(1) Detection of mouse IL-10 protein in the culture supernatant of recombinant B. breve ATCC 15700 The expression cassette of Example 1 for expression of IL-10 secretion is a species other than B. longum belonging to the genus Bifidobacterium We verified whether we have high ability. Using B. breve ATCC 15700, the expression cassettes F and H in Table 1 were observed by Western blotting as in Example 1. As a result, as shown in FIG. 6, secretory expression as high as that in B. longum was also observed in B. breve.

(2) Detection of mouse IL-10 protein in the culture supernatant of recombinant B. breve ATCC 15700 When each band on the Western blot image was analyzed in the same manner as in Example 1, the culture was performed as shown in Table 3. The IL-10 concentration in the supernatant was calculated.

From the above results, the expression cassettes F and H shown in Table 1 are not limited to B. longum, but also highly secreted expression of IL-10 in B. breve, another species of Bifidobacterium. It was shown to have the ability.

Recombinant B. longum 105-A that secretes and expresses mouse IL-10 with pTB4 vectors
(1) Construction of pTB4 system vector for mouse IL-10 secretion expression The expression cassette of Example 1 for IL-10 secretion expression has high ability even in Bifidobacterium plasmid system vectors other than the pTB6 plasmid of Example 1. We verified whether we have. Bifidobacterium plasmid replicon is a pTB4-based pBSM4 (formerly ps423 obtained from Professor Yasumasa Kano, Kyoto Pharmaceutical University, Yasumasa Kano, 6 others, 2008 Annual Meeting of the Japanese Society for Lactic Acid Bacteria, General Lecture 6) (MSC) An expression cassette F which is the expression cassette having the highest biological activity in the supernatant in Table 1 of Example 1 between HindIII and NotI of the multicloning site (MSC) of FIG. 2A) Inserted.

(2) Detection of mouse IL-10 protein in culture supernatant of recombinant B. longum 105-A For secretion expression of IL-10 in expression cassette F, pBSM4 was used by Western blotting as in Example 1. The four transformant clones were compared with transformants using pKKT427. As clearly shown in FIG. 7, when pTB4 pBSM4 was used, the amount of secretory expression was almost the same as when pTB6 pKKT427 was used.

From the above results, it can be seen that the result of the expression cassette of the present invention is not limited to the case of using the pTB6 pKKT427 vector. This is also shown in Example 8 as a secretory expression of TRAIL / Apo2-L.

Recombinant B. longum 105-A that secretes and expresses murine IL-10 using a post-signal sequence of BL1181 of various lengths
In the combination of the expression cassette F in Table 1 of Example 1, that is, the promoter region of cbaH, the signal sequence of BL1181 and the insertion sequence after the signal sequence of BL1181, the length of the insertion sequence after the signal sequence was changed, The amount of secretory expression was examined.

Following the signal sequence of the BL1181 gene product, 0, 1, 2, 3, 6, 9, 12, 16 consecutive amino acids counting immediately after the signal sequence are between the signal sequence of the BL1181 gene product and the BamHI site. An expression cassette was constructed by artificially synthesizing the HindIII site to the BamHI site by PCR by changing the BamHI side primer.

As in Example 1, mIL-10１C149Y was secreted and expressed in B.Blongum 105-A using pKKT427. The culture supernatant was subjected to buffer exchange with PBS / 0.1% BSA by gel filtration using a NAP column, and secreted mouse IL-10 protein was detected by Western blotting. FIG. 8 shows a Western blot image of IL-10 in the buffer exchanged solution. As shown here, the effects in the case of 1, 2, 3, 6, 9, 12, and 16 post-signal sequence insertion sequences are clearly shown as compared to the case where there is no post-signal sequence insertion sequence.

As in Example 1, the biological activity of secreted mouse IL-10 was measured by a proliferation assay using the mouse mast cell line MC / 9. The assay results for the buffer exchanged solution were as shown in FIG. As shown here, the effect in the case where the insertion sequence after the signal sequence is 1, 2, 3, 6, 9, 12, 16 amino acids is clearly shown as compared with the case where there is no insertion sequence after the signal sequence. Yes.

From the above results, when using the sequence following the signal sequence of the BL1181 gene product according to the present invention, the effect of remarkably promoting secretory expression when the length of the inserted sequence after the signal sequence is 1 to 16 amino acids Was shown to have.

B. longum 105-A that secretes and expresses human IL-2
(1) Construction of human IL-2 secretion expression vector An expression cassette was inserted between HindIII and NotI of the multicloning site of pKKT427 (FIG. 1). The expression cassette is ligated in the order of HindIII, promoter region, signal sequence, post-signal sequence insertion sequence, BamHI site, human IL-2 cDNA, stop codon, SpeI site, terminator, and NotI site (FIG. 3). Therefore, the amino acid GS encoded by BamHI's GGATCC is inserted into the expressed protein. In addition, the human IL-2 cDNA used here was obtained by removing the original signal sequence, and a mutant (hIL-2 C145S) from which cysteine unnecessary for activity was removed (Wang A, et al. Science. 1984). 224 (4656): 1431-3.) (SEQ ID NO: 16). As the terminator sequence, SEQ ID NO: 12 was used in which a single base mutation was added to the SpeI site originally possessed by the sequence based on the terminator of the hup gene of B. longum ATCC 15707. Table 4 shows combinations of expression cassettes up to the BamHI site.

(2) Detection of human IL-2 protein in culture supernatant of recombinant B. longum 105-A In the same manner as in Example 1, Western blotting analysis of the culture supernatant of the fungus was performed. Anti-human IL-2 polyclonal antibody (R & D Systems) was used as the primary antibody. Commercially available recombinant hIL-2 C145S (R & D Systems) was used as a positive control. As a negative control (Ctrl), a culture supernatant of B. longum 105-A transformed with pKKT427 without an expression cassette was used. FIG. 10 shows a Western blot image. As clearly shown here, a band was detected at the expected molecular weight, and the order of expression was A <C <B <E <D.

When each band on the Western blot image was analyzed in the same manner as in Example 1, the IL-2 concentration in the culture supernatant was calculated as shown in Table 5. To the best of our knowledge, this is the first report of IL-2 expression in Bifidobacterium.

(3) Measurement of biological activity of human IL-2 in the culture supernatant of recombinant B. longum 105-A The culture supernatant was determined by proliferation assay using mouse cytotoxic T cell line CTLL-2 (ATCC TIB-214). The biological activity of middle human IL-2 was measured. First, the culture supernatant was buffer exchanged with PBS / 0.1% BSA by gel filtration using a NAP column (GE Healthcare). CTLL-2 cells were seeded at 1 × 10 ⁴ cells / well in a 96-well plate. The buffer exchanged solution was diluted with the same buffer. The diluted solution was added to each well in a volume of 10% of the cell culture solution so that the final concentration of IL-2 in the CTLL-2 cell culture solution was 0.01 to 10 ng / ml. The cells were cultured for 24 hours and assayed using CellTiter-Glo (Promega). As a negative control (Ctrl), a culture supernatant of B. longum 105-A transformed with pKKT427 without an expression cassette was used. Commercially available recombinant hIL-2 C145S (R & D Systems) was used as a positive control.

The results of the assay were as shown in FIGS. 11A and B. The specific activity for the commercially available IL-2 protein is as shown in Table 6 and was not inferior to that of the commercially available IL-2 protein. In addition, the insertion sequence after the BL1181 signal sequence did not cause a decrease in activity.

From the above results, the combination of the promoter region of B. breve hup gene or B. longum cbaH, BL1181 signal sequence and BL1181 signal sequence post-insertion sequence is particularly excellent in IL-2 secretion expression. It was shown that.

B. longum 105-A secretes and expresses human IL-4 antagonistic mutant
(1) Construction of Human IL-4 Antagonist Mutant Secretion Expression Vector An expression cassette was inserted between HindIII and NotI of the multicloning site of pKKT427 (FIG. 1). The expression cassette is ligated in the order of HindIII, promoter region, signal sequence, post-signal sequence insertion sequence, BamHI site, human IL-4 antagonist mutant cDNA, stop codon, SpeI site, terminator, and NotI site (FIG. 3). ). Therefore, the amino acid GS encoded by BamHI's GGATCC is inserted into the expressed protein. In addition, the human IL-4 antagonistic mutant cDNA used here is a mutant (hIL4 T13D / R121D / Y124D) in which three amino acids are substituted, excluding the original signal sequence of human IL-4 (US Patent No. 6,028,176) (SEQ ID NO: 17). As the terminator sequence, SEQ ID NO: 12 was used in which a single base mutation was added to the SpeI site originally possessed by the sequence based on the terminator of the hup gene of B. longum ATCC 15707. Table 7 shows combinations of expression cassettes up to the BamHI site.

(2) Detection of human IL-4 antagonistic mutant protein in the culture supernatant of recombinant B. longum 105-A In the same manner as in Example 1, western blotting analysis of bacterial culture supernatant was performed. Anti-human IL-4 antibody (R & D Systems) was used as the primary antibody. As a negative control, a culture supernatant of B. longum 105-A transformed with pKKT427 without an expression cassette was used (Ctrl). Commercially available recombinant hIL-4 (rhIL4) (R & D Systems) was used as a positive control.

FIG. 12 shows a Western blot image. The expression of IL-4 antagonistic mutant was not observed in the combination B of the cbaH promoter region and the BL1181 signal sequence useful in IL-10 (Example 1) and IL-2 (Example 5). On the other hand, in the combination C of the rplJ promoter region and the BL1181 signal sequence, secretory expression was observed at the expected molecular weight. When the # 1 band of C on the Western blot image was analyzed in the same manner as in Example 1, the concentration in the supernatant was 49 ng / ml. To the best of our knowledge, this is the first report of IL-4 antagonistic mutant expression in Bifidobacterium.

From the above results, it was shown that the combination of the rplJ promoter region and the BL1181 signal sequence is excellent in the secretion expression of IL-4 antagonistic mutants.

B. longum 105-A that secretes and expresses human TRAIL / Apo2-L
(1) Construction of human TRAIL / Apo2-L secretion expression vector An expression cassette was inserted between HindIII and NotI of the multicloning site of pKKT427 (FIG. 1). The expression cassette is linked in the order of HindIII, promoter region, signal sequence, post-signal sequence insertion sequence, BamHI site, human TRAIL / Apo2-L cDNA, stop codon, SpeI site, terminator, and NotI site (FIG. 3). . Therefore, the amino acid GS encoded by BamHI's GGATCC is inserted into the expressed protein. The human TRAIL / Apo2-L cDNA used here is a solubilized form of amino acids 114-281 (Ashkenazi A, et al. J Clin Invest. 1999. 104 (2): 155-62.) SEQ ID NO: 18). As the terminator sequence, SEQ ID NO: 12 was used in which a single base mutation was added to the SpeI site originally possessed by the sequence based on the terminator of the hup gene of B. longum ATCC 15707. Table 8 shows combinations of expression cassettes up to the BamHI site.

(2) Detection of human TRAIL / Apo2-L protein in the culture supernatant of recombinant B. longum 105-A In the same manner as in Example 1, Western blotting analysis of the bacterial culture supernatant was performed. Biotinylated anti-human TRAIL / Apo2-L antibody (R & D Systems) was used as the primary antibody. A commercially available recombinant solubilized hTRAIL / Apo2-L (PeproTech) was used as a positive control. FIG. 13 shows a Western blot image of each culture time with the expression cassette A in Table 8. Some degradation products are seen at the incubation time of 9 hours. The concentration of TRAIL / Apo2-L in the culture supernatant at a culture time of 6 hours was calculated to be 1.08 μg / ml. Hereinafter, the culture supernatant with a culture time of 6 hours was analyzed.

FIG. 14 shows Western blot images of expression cassettes A to D in Table 8. FIG. 15 shows Western blot images of expression cassettes A and E in Table 8. 14 and 15, Ctrl indicates the culture supernatant of B. longum 105-A transformed with pKKT427 that does not contain the expression cassette. Table 9 shows the ratio of the TRAIL / Apo2-L concentration in the culture supernatant in each expression cassette, where the concentration of TRAIL / Apo2-L in the culture supernatant in the expression cassette A is 100.

Many combinations of other promoter regions, signal sequences and post-signal sequence insertion sequences were also tried. However, the amount of secretory expression exceeding the promoter region of hup and the promoter region of cabH was not obtained. In addition, expression cassettes that are superior in different genera, such as a combination of the P1 promoter of Lactococcus lactis and the signal sequence of usp45 (Steidler L, et al. Science. 2000. 289 (5483): 1352-5.) In bacteria, as long as it was tried, the combination of A to E was not far (data not shown).

(3) Measurement of biological activity of human TRAIL / Apo2-L in the culture supernatant of recombinant B. longum 105-A. Apoptosis assay using human colorectal adenocarcinoma cell line COLO 205 (ATCC CCL-222) The biological activity of Kiyonaka human TRAIL / Apo2-L was measured. COLO 205 cells were seeded at 1 × 10 ⁴ cells / well in a 96-well plate and allowed to adhere overnight. Then, filter-filtered recombinant B. longum 105-A culture supernatant or a solution obtained by diluting the filtered culture supernatant with MRS-HEPES medium is added to each well in a volume of 10% of COLO 205 cell culture solution. Added. The final concentration of the culture supernatant of the added recombinant B. longum 105-A in the COLO 205 cell culture solution was adjusted to 0 to 10% (v / v). Cultured for 24 hours and assayed using CellTiter-Glo (Promega, Madison, Wis.). As a negative control (Ctrl), a culture supernatant of B. longum 105-A transformed with pKKT427 without an expression cassette was used. As a positive control, commercially available recombinant solubilized hTRAIL / Apo2-L (PeproTech) was used.

The results of the assay from expression cassettes A to D were as shown in FIG. TRAIL / Apo2-L secreted by the signal sequence of amyB had biological activity, but unexpectedly, the one secreted by the signal sequence of BL1181 lost its biological activity.

From the above results, in secretory expression of TRAIL / Apo2-L in Bifidobacterium, the promoter region of the cbaH gene or the promoter region of the hup gene, more preferably the promoter region of the hup gene of B. breve, and amyB It was shown that the combination of the signal sequences was particularly excellent.

B. longum 105-A that secretes and expresses human TRAIL / Apo2-L with pTB4 vectors
(1) Construction of pTB4 vector for secretion expression of human TRAIL / Apo2-L Bifidobacterium plasmid vector other than the pTB6 plasmid of Example 7 is used as the expression cassette of Example 7 for TRAIL / Apo2-L secretion expression. We verified whether we have high ability. Bifidobacterium plasmid replicon is pBSM11 pBSM11 (former pBS423 obtained from Prof. Yasumasa Kano, Kyoto University of Pharmacy, 6 others, 2008 Annual Meeting of the Japanese Society for Lactic Acid Bacteria, General Lecture 6) (MSC) Inserted between the HindIII and NotI sites of the multiple cloning site of FIG. 2B), the expression cassette E, which is the expression cassette with the highest biological activity in the supernatant in Table 8 of Example 7, was inserted. .

(2) Detection of human TRAIL / Apo2-L protein in the culture supernatant of recombinant B. longum 105-A Secretory expression of TRAIL / Apo2-L in expression cassette E was performed by Western blotting in the same manner as in Example 1. The transformant using pBSM11 was compared with the transformant using pKKT427. As clearly shown in FIG. 17, when pTB4 pBSM11 was used, the amount of secretory expression was almost the same as when pTB6 pKKT427 was used.

(3) Bioactivity measurement of human TRAIL / Apo2-L in the culture supernatant of recombinant B. longum 105-A Apoptosis assay using human colorectal adenocarcinoma cell line COLO 205 (ATCC CCL-222) The biological activity of Kiyonaka human TRAIL / Apo2-L was measured. First, the culture supernatant was buffer exchanged with RPMI medium by gel filtration using a NAP column (GE Healthcare). COLO 205 cells were seeded at 1 × 10 ⁴ cells / well in a 96-well plate and allowed to adhere overnight. The buffer-exchanged solution was diluted with the same buffer so as to have each concentration. The dilution was added to each well in a volume of 10% of COLO 205 cell culture. Cultured for 24 hours and assayed using CellTiter-Glo (Promega, Madison, Wis.). As a negative control (Ctrl), a culture supernatant of B. longum 105-A transformed with pKKT427 without an expression cassette was used. A commercially available recombinant solubilized hTRAIL / Apo2-L (PeproTech) was used as a positive control. The specific activity of cytotoxicity when using pTBSM11 of pTB4 system when the cytotoxicity (Cytotoxicity) when using pKKT427 of pTB6 system is 100 is 97, and the organism of TRAIL / Apo2-L in the culture supernatant The activity was almost equivalent.

From the above results, it can be seen that the result of the expression cassette of the present invention is not limited to the case of using the pTB6 pKKT427 vector. This is also shown in Example 3 as a case of IL-10 secretion expression.

B. longum 105-A that secretes and expresses human IFN-β
(1) Construction of human IFN-β secretion expression vector An expression cassette was inserted between HindIII and NotI of the multicloning site of pKKT427 (FIG. 1). The expression cassette is ligated in the order of HindIII, promoter region, signal sequence, post-signal sequence insertion sequence, BamHI site, human IFN-β cDNA, stop codon, SpeI site, terminator, and NotI site (FIG. 3). Therefore, the amino acid GS encoded by BamHI's GGATCC is inserted into the expressed protein. Further, the human IFN-β cDNA used here is the one excluding the original signal sequence (SEQ ID NO: 19). As the terminator sequence, SEQ ID NO: 12 was used in which a single base mutation was added to the SpeI site originally possessed by the sequence based on the terminator of the hup gene of B. longum ATCC 15707. Table 10 shows combinations of expression cassettes up to the BamHI site.

(2) Detection of human IFN-β protein in culture supernatant of recombinant B. longum 105-A In the same manner as in Example 1, western blotting analysis of the culture supernatant of the bacteria was performed. However, here, the culture supernatant and cells after 8 hours of culture were collected. Biotinylated anti-human IFN-β goat polyclonal IgG (R & D Systems) was used as the primary antibody. Commercially available recombinant hIFN-β1a (PBL InterferonSource, Piscataway, NJ) was used as a positive control. As a negative control (Ctrl), a culture supernatant of B. longum 105-A transformed with pKKT427 without an expression cassette was used. FIG. 18 shows a Western blot image. Only when the insertion sequence after the signal sequence was used (expression cassettes B to E), secretory expression was observed mainly at the expected molecular weight.

When the predicted molecular weight band was analyzed in the same manner as in Example 1, the IFN-β concentration in the culture supernatant was calculated as shown in Table 11. To the best of our knowledge, this is the first report of type I IFN expression in Bifidobacterium.

(3) Measurement of biological activity of human IFN-β in the culture supernatant of recombinant B. longum 105-A In the culture supernatant of B and D by reporter gene assay using iLite alphabeta Kit (Neutekbio, Galway, Ireland) The biological activity of human IFN-β was measured. First, the culture supernatant was buffer exchanged with DMEM medium by gel filtration using a NAP column (GE Healthcare). The results of the assay were as shown in FIGS. 19A and B. The calculated interferon activity in the culture supernatant is as follows: # 1 for B is 5.6 x 10 ⁵ IU / ml, # 2 is 2.4 x 10 ⁵ IU / ml, # 1 for D is 1.9 x 10 ⁴ IU / ml, # 2 was 1.3 × 10 ⁴ IU / ml. From this result, it can be seen that the specific activity of interferon is superior to that of LEISS using the BL1181 post-signal sequence insertion sequence.

From the above results, by adding the insertion sequence after the signal sequence, IFN-β, which could not detect secretory expression without addition, could be expressed at a high level. The effect is clear. Regarding the biological activity of IFN, the signal sequence inserted after BL1181 was superior to LEISS.

B. longum 105-A that secretes and expresses human R-spondin 2
(1) Construction of human R-spondin 2 secretion expression vector An expression cassette was inserted between HindIII and NotI of the multicloning site of pKKT427 (FIG. 1). The expression cassette is ligated in the order of HindIII, promoter region, signal sequence, post-signal sequence insertion sequence, BamHI site, human R-spondin 2 dC cDNA, tag sequence, stop codon, SpeI site, terminator, and NotI site ( FIG. 3). Therefore, the amino acid GS encoded by BamHI's GGATCC is inserted into the expressed protein. The human R-spondin 2 cDNA used here is a dC type (R-spondin 2 dC) excluding the signal sequence of human R-spondin 2 and the sequence encoding the C-terminal highly charged amino acid region. (SEQ ID NO: 20; WO2007 / 013666). The tag sequence is a DNA encoding an artificial sequence including a V5 tag and a polyhistidine tag (SEQ ID NO: 21). As the terminator sequence, SEQ ID NO: 12 was used in which a single base mutation was added to the SpeI site originally possessed by the sequence based on the terminator of the hup gene of B. longum ATCC 15707. Table 12 shows combinations of expression cassettes up to the BamHI site.

(2) Detection of human R-spondin 2 protein in the culture supernatant of recombinant B. longum 105-A In the same manner as in Example 1, Western blotting analysis of the culture supernatant of the bacteria was performed. However, here, the culture supernatant and cells after 8 hours of culture were collected. As an antibody for detection, HRP-labeled anti-polyhistidine mouse monoclonal antibody (R & D Systems) was used. As a positive control, purified recombinant R-spondin 2 dC protein (WO2007 / 013666) was used. As a negative control (Ctrl), a culture supernatant of B. longum 105-A transformed with pKKT427 without an expression cassette was used. FIG. 20 shows a Western blot image. Although there was considerable survival in the cells, secretory expression was observed at the expected molecular weight.

When the predicted molecular weight band was analyzed in the same manner as in Example 1, the concentration of R-spondin 2 in the culture supernatant was calculated as shown in Table 13. From this result, it can be seen that the signal sequence post-insertion sequence LEISS is superior to BL1181 as the amount of protein to be secreted. To the best of our knowledge, this is the first report on the expression of R-spondins in Bifidobacterium.

From the above results, it was found that R-spondin 2 can be secreted and expressed in Bifidobacterium according to the present invention.

In vivo tumor growth inhibitory effect of human TRAIL / Apo2-L expressing Bifidobacterium (1)
Seven-week-old female BALB / c nude mice (Charles River Laboratories Japan, Yokohama, Japan) were transplanted subcutaneously with 5 x 10 ⁶ COLO 205 cells (ATCC CCL-222), resulting in well-established tumors . The tumor tissue was collected and cut to obtain approximately 3 mm ³ square COLO 205 tumor pieces. The tumor pieces were subcutaneously transplanted into 7-week-old female BALB / c nude mice to obtain tumor-bearing mice. Mice were grouped when tumor size reached an average of 243 mm ³ (day 0) (n = 8). Tumor volume was normalized to 100% on Day 0. On Day 1, 4, 8 and 11, 50 μl of approximately 2 × 10 ⁸ cfu bacteria were administered intratumorally using a syringe. In the other two groups, the same volume of vehicle (PBS) alone or recombinant B. longum 105-A transformed with empty pKKT427 containing no expression cassette was administered intratumorally. The Bifidobacterium used was prepared by centrifugation and resuspension in PBS. All mice received 1 ml of 20% lactulose (Sigma-Aldrich, St Louis, MO) 3 times per week intraperitoneally to supplement the body with Bifidobacterium.

FIG. 21 shows solubilized human TRAIL with hup promoter region and amyB signal sequence compared to the negative control recombinant B. longum 105-A (Ctrl) transformed with empty pKKT427 without the expression cassette. B. を longum 105-A (A in Table 8) (TRAIL) secreting / expressing / Apo2-L clearly suppressed the growth of established tumors. Data are displayed as mean + SEM.

FIG. 22 shows the weight change of the same experiment. All groups showed an increase or decrease in body weight that was thought to be due to food exchange, but the body weight reduction was small in the TRAIL / Apo2-L expressing bacteria administration group.

In vivo tumor growth inhibitory effect of human TRAIL / Apo2-L expressing Bifidobacterium (2)
Seven-week-old female BALB / c nude mice (Charles River Laboratories Japan, Yokohama, Japan) were transplanted subcutaneously with 5 × 10 ⁶ COLO 205 cells to obtain well-established tumors. The tumor tissue was collected and cut to obtain approximately 3 mm ³ square COLO 205 tumor pieces. The tumor pieces were subcutaneously transplanted into 7-week-old female BALB / c nude mice to obtain tumor-bearing mice. Mice were grouped when tumor size reached an average of 444 mm ³ (day 0). Tumor volume was normalized to 100% on Day 0. On day 0, 50 μl of approximately 2 × 10 ⁸ cfu bacteria were administered intratumorally using a syringe. The Bifidobacterium used was prepared by centrifugation and resuspension in PBS. Mice were given 1 ml of 20% lactulose (Sigma-Aldrich, St Louis, MO) intraperitoneally three times a week to supplement the body with Bifidobacterium.

FIG. 23 secretes and expresses solubilized human TRAIL / Apo2-L with the cbaH promoter region and amyB signal sequence, as compared with the negative control recombinant B. longum 105-A (Ctrl) (n = 4) B. longum 105-A (C in Table 8) (TRAIL) (n = 5) showed that the growth of established tumors was suppressed by only a single dose. Data are displayed as mean + SEM.

From the results of Examples 11 and 12, it was shown that Bifidobacterium expressing solubilized TRAIL may be useful for tumor therapy.

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

The present invention provides an expression cassette capable of efficiently secreting and expressing a polypeptide in Bifidobacterium. Such an expression cassette is useful for producing a recombinant microorganism that secretes and expresses a polypeptide. Such a recombinant microorganism can be used as a pharmaceutical or a component constituting a pharmaceutical for treating and / or preventing diseases of animals including humans. And it can provide a new treatment and / or prevention method for the disease. The expression cassette and the recombinant microorganism can also be used for industrial polypeptide production.

SEQ ID NO: 8: synthetic peptide (insertion sequence after signal sequence)
SEQ ID NO: 9: synthetic peptide (insert sequence after signal sequence)
SEQ ID NO: 10: synthetic DNA (insert sequence after signal sequence)
SEQ ID NO: 11: synthetic DNA (sequence inserted after signal sequence)
SEQ ID NO: 12: synthetic terminator sequence SEQ ID NO: 14: synthetic DNA
SEQ ID NO: 15: synthetic DNA (mouse interleukin 10 mature peptide C149Y mutant cDNA)
SEQ ID NO: 16: synthetic DNA (human interleukin 2 mature peptide C145S mutant cDNA)
SEQ ID NO: 17: Synthetic DNA (human interleukin 4 mature peptide T13D / R121D / Y124D mutant cDNA)
SEQ ID NO: 20: Synthetic DNA (human dC type R-spondin2 mature peptide cDNA)
SEQ ID NO: 21: synthetic DNA (tag sequence)

Claims (26)

An expression cassette comprising the following (i), (ii) and (iii):
(I) a promoter selected from the group consisting of a promoter of a cbaH gene derived from a Bifidobacterium bacterium, a promoter of an rplJ gene derived from a Bifidobacterium bacterium, and a promoter of a hup gene derived from a Bifidobacterium bacterium,
(Ii) DNA encoding a signal sequence selected from the group consisting of the signal sequence of the BL1181 gene product derived from Bifidobacterium and the signal sequence of the amyB gene product derived from Bifidobacterium,
(Iii) DNA encoding a polypeptide or a cloning site for inserting the DNA The expression cassette according to claim 1, wherein DNA encoding a polypeptide is inserted into (iii). The DNA encoding 1 to 20 consecutive amino acids following the signal sequence of the BL1181 gene product derived from Bifidobacterium is linked between (ii) and (iii), 2. The expression cassette according to 2. The expression cassette according to claim 1 or 2, wherein DNA encoding the amino acid sequence represented by SEQ ID NO: 9 is linked between (ii) and (iii). An expression cassette comprising the following (i), (ii), (iii) and (iv):
(I) a promoter of a gene derived from a genus Bifidobacterium,
(Ii) DNA encoding a signal sequence of a gene product derived from a genus Bifidobacterium,
(Iii) DNA encoding 1 to 20 consecutive amino acids following the signal sequence of a gene product derived from a genus Bifidobacterium,
(Iv) DNA encoding a polypeptide or a cloning site for inserting the DNA 6. The expression cassette according to claim 5, wherein DNA encoding the polypeptide is inserted into (iv). The promoter is selected from the group consisting of a promoter of a cbaH gene derived from a Bifidobacterium bacterium, a promoter of an rplJ gene derived from a Bifidobacterium bacterium, and a promoter of a hup gene derived from a Bifidobacterium bacterium. Item 7. The expression cassette according to Item 5 or 6. The polypeptides include interleukin-10 (IL-10), interleukin-2 (IL-2), TRAIL / Apo2-L, type I interferon, interleukin-4 (IL-4) antagonistic mutant, and R- The expression cassette according to any one of claims 1 to 7, which is selected from the group consisting of spondins. The cbaH gene promoter derived from Bifidobacterium longum, DNA encoding the signal sequence of the BL1181 gene product derived from Bifidobacterium longum, and DNA encoding IL-10. Expression cassette. A cbaH gene promoter derived from Bifidobacterium longum or a hup gene promoter derived from Bifidobacterium longum, DNA encoding the signal sequence of the BL1181 gene product derived from Bifidobacterium longum, and Bifidobacterium longum The expression cassette according to claim 8, comprising DNA encoding 1 to 20 contiguous amino acids or DNA encoding LEISSTCDA following the signal sequence of BL1181 gene product derived from longum, and DNA encoding IL-10. . Hup gene promoter from Bifidobacterium breve or cbaH gene promoter from Bifidobacterium longum, DNA encoding the signal sequence of BL1181 gene product from Bifidobacterium longum, and IL-2 coding The expression cassette according to claim 8, which contains DNA to be expressed. Hup gene promoter from Bifidobacterium breve or cbaH gene promoter from Bifidobacterium longum, DNA encoding the signal sequence of BL1181 gene product from Bifidobacterium longum, Bifidobacterium The expression cassette according to claim 8, comprising DNA encoding 1 to 20 contiguous amino acids following the signal sequence of the BL1181 gene product derived from longum and DNA encoding IL-2. An rplJ gene promoter derived from Bifidobacterium longum, a DNA encoding a signal sequence of a BL1181 gene product derived from Bifidobacterium longum, and a DNA encoding an IL-4 antagonistic mutant 8. The expression cassette according to 8. Hup gene promoter from Bifidobacterium breve or hup gene promoter from Bifidobacterium longum, DNA encoding the signal sequence of the amyB gene product from Bifidobacterium adrecentis, and TRAIL / Apo2 The expression cassette according to claim 8, comprising DNA encoding -L. Hup gene promoter from Bifidobacterium longum or cbaH gene promoter from Bifidobacterium longum, DNA encoding the signal sequence of BL1181 gene product from Bifidobacterium longum, and type I interferon The expression cassette according to claim 8, which contains DNA to be expressed. Hup gene promoter from Bifidobacterium longum or cbaH gene promoter from Bifidobacterium longum, DNA encoding the signal sequence of BL1181 gene product from Bifidobacterium longum, and Bifidobacterium longum The expression cassette according to claim 8, comprising DNA encoding 1 to 20 contiguous amino acids or DNA encoding LEISSTCDA following the signal sequence of BL1181 gene product derived from longum, and DNA encoding type I interferon. . The cbaH gene promoter derived from Bifidobacterium longum, DNA encoding the signal sequence of the BL1181 gene product derived from Bifidobacterium longum, and DNA encoding R-spondins Expression cassette. The cbaH gene promoter from Bifidobacterium longum, the DNA encoding the signal sequence of the BL1181 gene product from Bifidobacterium longum, and the signal sequence of the BL1181 gene product from Bifidobacterium longum 1 The expression cassette according to claim 8, comprising DNA encoding ˜20 consecutive amino acids or DNA encoding LEISSTCDA and DNA encoding R-spondins. The expression cassette according to any one of claims 1 to 18, which is used for secreting and expressing a polypeptide in a bacterium belonging to the genus Bifidobacterium. An expression vector containing the expression cassette according to any one of claims 2 to 4 and 6 to 19. A recombinant microorganism comprising the expression cassette according to any one of claims 2 to 4 and 6 to 19 or the expression vector according to claim 20. The microorganism according to claim 21, wherein the microorganism is a bacterium belonging to the genus Bifidobacterium. A pharmaceutical composition comprising the microorganism according to claim 21 or 22. 24. A pharmaceutical composition according to claim 23 for treating a manic disease or disorder. 25. The pharmaceutical composition according to claim 24, wherein the hemorrhoid disease or disorder is inflammatory bowel disease or tumor disease. A recombinant microorganism containing the expression cassette according to any one of claims 2 to 4 and 6 to 19 or the expression vector according to claim 20 is cultured, and the polypeptide is collected from the resulting culture. A method for producing a polypeptide.

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