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WO1993014203A1 - Lymphotoxines, vecteur d'expression pour lymphotoxines, et production de lymphotoxines au moyen de ce vecteur - Google Patents

Lymphotoxines, vecteur d'expression pour lymphotoxines, et production de lymphotoxines au moyen de ce vecteur Download PDF

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Publication number
WO1993014203A1
WO1993014203A1 PCT/JP1992/000043 JP9200043W WO9314203A1 WO 1993014203 A1 WO1993014203 A1 WO 1993014203A1 JP 9200043 W JP9200043 W JP 9200043W WO 9314203 A1 WO9314203 A1 WO 9314203A1
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Prior art keywords
gene
vector
lymphotoxins
lymphotoxin
protein
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Ceased
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PCT/JP1992/000043
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English (en)
Japanese (ja)
Inventor
Toshimi Morita
Kumiko Yano
Kyouko Yamamoto
Hideki Takagi
Toshiaki Fujihashi
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Tsumura and Co
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Tsumura and Co
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Priority to PCT/JP1992/000043 priority Critical patent/WO1993014203A1/fr
Publication of WO1993014203A1 publication Critical patent/WO1993014203A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Lymphotoxins their expression vectors, and methods for producing lymphotoxins using the same
  • the present invention relates to a vector for advantageously expressing a lymphotoxin, a method for producing a lymphotoxin using the vector, and a lymphotoxin produced by the vector.
  • TNF tumor necrosis factor
  • lymphotoxin lymphotoxin
  • antitumor peptides have been known as antitumor peptides, and their clinical use has been studied.
  • TNF has problems of side effects such as lowering of blood pressure and fever at the time of administration, and is considered to be difficult to apply to actual treatment.
  • lymphotoxins and lymphotoxins such as a lymphotoxin-like peptide having high homology thereto have no side effects unlike TNF, but have problems in their production.
  • lymphotoxins are mainly produced by genetic recombination, but their expression levels are low, resulting in poor production efficiency and securing sufficient amounts of lymphotoxins for actual treatment. It was difficult at present.
  • lymphotoxins which had been expressed very little in the past, could be efficiently expressed, and completed the present invention.
  • an object of the present invention is to have an SD region 13 bases upstream of the start codon, a base sequence of 110 region is TT AACT, a base sequence of 135 region is TTGACA, and Expression of lymphotoxins with a size of 2.7-3.1 kb excluding genes encoding
  • the idea is to submit a vector.
  • Another object of the present invention is to provide a method for producing lymphotoxins, which comprises transforming a host microorganism with the vector described above and culturing the transformed microorganism.
  • Yet another object of the present invention is to introduce a peptide having high homology to a novel natural lymphotoxin advantageously prepared by the method of the present invention.
  • FIG. 1 to FIG. 3 are drawings showing steps for constructing the vector of the present invention.
  • FIG. 4 is a drawing showing gene 11 and a peptide having high homology to the natural lymphotoxin encoded by the gene (hereinafter, may be referred to as the peptide of the present invention).
  • the vector of the present invention uses a promoter in which the nucleotide sequence of the 10 region is TTAACT and the nucleotide sequence of the 135 region is TTGACA, and uses a promoter between the SD region (ribosome binding site) and the initiation codon (ATG). 13 bases, to which a gene coding for a lymphotoxin (hereinafter abbreviated as “LT”) (hereinafter abbreviated as “LT structural gene”) is ligated, and a vector excluding the LT structural gene is ligated. It can be constructed by setting the base length to 2.7 to 3.1 kb.
  • the vector of the present invention comprises a base sequence encoding the first half of the promoter (gene 1), the second half of the promoter including the SD region and the start codon, and the first half of the LT gene.
  • an expression vector (gene 5) It can be built by performing a game.
  • the distance between the SD region and the start codon can be reduced to 13 bases by using the chemically synthesized gene 2.
  • the expression vector to be ligated requires that the constructed vector of the present invention has a base length of 2.7 to 3.1 kb excluding the LT structural gene. Yes, for example, by using PUC-based PUC, PTZ, Bluescript, etc., the demand can be satisfied.
  • the LT structural gene used in the vector of the present invention is a natural lymphotoxin gene
  • a part thereof is modified by utilizing the natural lymphotoxin gene or completely synthesized by organic synthesis. It may be a gene encoding an artificially produced peptide with high homology to natural lymphotoxin.
  • the LT structural gene of the LT expression vector obtained by the method of FIG. 1 can be exchanged for another LT structural gene. That is, a gene (gene 6) encoding most of the promoter is cut out from the LT expression vector prepared by the method shown in FIG. 1 and cut out from this and another vector or synthesized. A gene (structural gene) that contains a gene (gene 8) encoding most of the LT structural gene, an SD region and an initiation codon (ATG), and is prepared so that this distance is 13 bases. 7 or 10) and an expression vector (gene 3) are ligated to construct the vector of the present invention.
  • a gene (gene 6) encoding most of the promoter is cut out from the LT expression vector prepared by the method shown in FIG. 1 and cut out from this and another vector or synthesized.
  • the method shown in FIG. 3 incorporates the LT structural gene of the LT expression vector obtained by the method of FIG. 1 into another vector as it is, and the number of copies, etc.
  • the vector of the present invention having a characteristic can be obtained.
  • the above method is one of the methods for preparing the LT expression vector of the present invention. It is an example and can be prepared by other methods.
  • a promoter that satisfies the condition that the nucleotide sequence of the ⁇ 10 region is TT AACT and the nucleotide sequence of the 135 region is TT GACA includes T rp pro It is also possible to incorporate the LT structural gene into a vector having the same rp promoter and remove the unnecessary base from the resulting vector to prepare the vector of the present invention.
  • the LT expression vector obtained as described above can be used to transform a host microorganism in accordance with a conventional method and culture the transformed microorganism to produce LT.
  • Transformants can be cultured in a conventional manner, using L-culture medium, M9 medium, M9-force amino acid medium, high phosphoric acid medium, high phosphoric acid-casamino acid medium, etc. Can be used.
  • 3-indoleacrylic acid to the medium.
  • the amount of ⁇ -indoleacrylic acid to be added is about 20 to about L O Og per 1 ml of the medium.
  • the microorganism In order to separate and purify the LT obtained by the culture from the culture, the microorganism is destroyed by, for example, sonication or the like, and then a supernatant is obtained by centrifugation or the like. It is carried out by subjecting to a purification method in which purification means such as dialysis, ion chromatography, hydrophobic chromatography, and affinity chromatography are combined.
  • trp promoter 1 a promoter portion whose base sequence in the 10 region is TT AACT, and a base sequence in the 35 region is TTGACA (hereinafter referred to as “trp promoter 1”), and an SD region and Of the LT structural gene (hereinafter abbreviated as "MLT”) prepared according to Reference Example (4) described later (Japanese Patent Application No. Hei.
  • the head (gene 2) was organically synthesized using a DNA synthesizer.
  • Gene 1 and gene 2 were synthesized using the following oligonucleotides (1) to (6) and the following oligonucleotides (7) to (14).
  • Pkk223-3-3 MLT DNA disclosed in Reference Example (5) was cut with a restriction enzyme EcoRI-Seal (Toyobo), and a DNA fragment of 495 bp was recovered.
  • the DNA was digested with N sil (NEB) and a DNA fragment (gene 3) of 431 bp was recovered.
  • the gene 5 was reacted with the genes 1, 2, 3 and 4 using T4 DNA ligase to obtain a plasmid (PBTrpMLT) having the MLT gene. Further, the HB101 strain for transformation which had been frozen and stored at 170 ° C. was thawed, and 20 ng of pBTrpMLT was added to the suspension 1001. After reaction on ice for 45 minutes, a heat shock was applied at 42 ° C for 90 seconds, and the mixture was cooled for 2 minutes. After adding SOC Medium 900 ⁇ 1 and culturing at 37 ° C for 1 hour, the suspension is spread on an LB agar plate containing 0.1 mg Zm1 ambicilin, and then incubated at 37 ° C.
  • transgenic plants having a vector (pB trp MLT) comprising genes 1, 2, 3, 4, and 5 were obtained.
  • DNA was isolated from this transformant, cut with the restriction enzyme EcoRI — Hindi !, and then electrophoresed on a 1% agarose gel. EtBr staining confirmed a fragment of 629 bp. did.
  • the expression level of MLT per total protein was measured with an LKB laser scanner (Pharmacia LKB). At culture conditions of 30 ° C, 37% of total protein and 37 e Under the culture conditions of C, expression of 25% of the MLT protein per total protein was confirmed. This corresponds to a protein mass of 40 to 80 mg per little under culture conditions at 30 ° C.
  • the obtained cells (33.7 g / wet) were suspended in 50 ml of 50 mM Tris-HCl (pH 8.0) containing 30 mM NaCl and 0.1 mM p-APMSF, and This cell suspension was passed through a high-pressure homogenizer (RANNIE) three times at 600 psi to destroy the cells. Further, the supernatant was obtained by removing the cell residue by centrifugation.
  • RANNIE high-pressure homogenizer
  • Polyethyleneimine was added to 500 ml of the obtained supernatant to a final concentration of 0.1%, and the mixture was stirred at 4 ° C for 10 minutes, centrifuged, and the nucleic acid was removed. Done. 480 ml of the supernatant was recovered, and ammonium sulfate was added to 50% saturation, followed by stirring at 4 eC for 6 hours, followed by centrifugation to obtain a crude protein as a precipitate.
  • This crude protein was dissolved in 45 ml of 5 mM phosphate buffer (pH 7.4), and dialyzed against the same buffer. After further centrifugation, the insoluble protein was removed, and 52 ml of the supernatant was recovered. Further, a heat treatment was performed at 50 ° C for 10 minutes to remove proteins to be precipitated. Prepare 50 ml of the recovered solution in advance The column was applied to a column of DE AE Sepharose Fast Flow (Pharmacia LKB) equilibrated with 5 mM phosphate buffer (pH 7.4), and 5 mM phosphate buffer (PH 7.4). Was eluted.
  • the eluate was purified by an ultrafilter (YM10 membrane) manufactured by Amicon, to obtain a concentrated solution of 130 ml.
  • the concentrated solution was dialyzed against 5 mM phosphate buffer (pH 7.0) to obtain a 132 ml protein solution.
  • the obtained protein solution was adsorbed onto a column of CM Sepharose Fast Flour (Pharmacia LKB II) equilibrated with 5 mM phosphate buffer (PH 7.0), After washing with a phosphate buffer (pH 7.0), elution was carried out with a stepwise concentration gradient with 5 mM phosphate buffer (pH 7.0) containing NaCl.
  • the protein purified by the present invention was recovered at a purity of 95% or more.
  • the pt r p M L T DNA obtained in Example 1 was replaced with a restriction enzyme BamHI.
  • a gene ( ⁇ 7) that encodes the head of LT was prepared while keeping the SD region and initiation codon separated by 13 bases.
  • 0.1 OD of each of the oligonucleotides (2) to (5) is dissolved in sterilized water 151, and T4 polynucleotide kinase 40 is dissolved.
  • the reaction was performed at C for 2 hours. After the reaction, 65.
  • the enzyme was inactivated by heat treatment with C for 10 minutes.
  • T4 DNA ligase 2000 units (Takara Shuzo), 1M DTT11, 10mM ATP10 ⁇ 1, and annealing ring buffer 71 were added, and the mixture was added. Then 16. The mixture was reacted for 16 hours with C, electrophoresed on a 10% PAGE gel, stained with EtBr, the target gene 7 was excised, eluted in an elution buffer, and desalted.
  • the nucleotide sequence of the obtained gene 7 is as follows.
  • T4 DNA ligase was generated for genes 6, 7, 8, and 9 to obtain a plasmid having the LT gene (pBTrpLT). Further, the HB101 strain for transformation, which had been frozen and stored at -70, was thawed, and 20 ng of pBTrpLT was added to the suspension lOOJl. After 45 minutes of ice-cooling, a heat shock was applied at 42 ° C for 90 seconds, followed by ice-cooling for 2 minutes. To this, add S0C medium 9001 and incubate at 37 for 1 hour.Pour the suspension onto LB agar plate containing O.lmgZml of ampicillin, and place it at 37 ° C.
  • ff-indoleacrylic acid (20 gZml) was added, and the cells were further cultured at 37 ° C for 16 hours. After completion of the culture, 1 ml of the bacterial cell fluid was transferred to a 1.5 ml centrifuge tube, and the cells were obtained by centrifugation. The cells were lysed in 200 1 SDSPAGE buffer, heat treated at 90 ° C for 5 minutes, and electrophoresed on 15% SDSPAGE. After the electrophoresis, the cells were stained with Kumashi Blue and then decolorized.
  • LKB laser scanner was used to measure the expression level of LT per total protein, and it was confirmed that 10% of LT protein was expressed per total protein under the culture condition of 37 ° C. It was done. This corresponds to a protein content of 10 to 20 mg per liter under the culture condition of 37 ° C.
  • the obtained bacterial cells (15, 95 g / wet) were added to 30 mM NaCl and
  • Polyethyleneimine is added to 500 ml of this crude extract to a final concentration of 0.1%, stirred at 10 minutes for 10 minutes, centrifuged, and nucleic acid is removed.
  • Collect 480 ml of the supernatant add ammonium sulfate to 50% saturation, and stir at 4 eC for 5 hours.
  • centrifugation was performed to obtain a crude protein as a precipitation source. This protein was dissolved in 45 ml of 5 mM phosphate buffer (PH 7.4) and dialyzed against the same buffer. After further centrifugation, insoluble proteins were removed, and 52 ml of the supernatant was recovered. Further, a heat treatment was performed at 50 ° C.
  • the eluate was concentrated using an ultrafilter (YM10 membrane) manufactured by Amicon, to obtain 130 ml of a concentrated solution.
  • This concentrated solution was dialyzed against 5 mM phosphoric acid buffer solution (PH 7.0) to obtain 132 ml of a protein solution.
  • the protein solution was adsorbed onto a column of CM Sepharose Fast Flow (Pharmacia LKB ⁇ ) equilibrated with 5 mM phosphate buffer (pH 7.0), and the solution was treated with 5 mM phosphate buffer (PH After washing with 7.0), elution was carried out with 5 mM phosphate buffer (pH 7.0) containing NaCl according to a stepwise concentration gradient.
  • the fraction containing the peptide was collected, dialyzed against a phosphate buffer, and the activity was determined by an L929 test, and the specific activity and the recovery in each purification step were determined.
  • the protein quantification was measured using a Protein Attay system manufactured by Piolado. Table 2 shows the results.
  • Purified protein 2 Cig was electrophoresed in 15% SDS PAGE, stained with Coomassie blue, decolorized, and tested for purity with an LKB laser scanner. As a result, the protein purified by the present invention was recovered with a purity of 95% or more.
  • TLT recombinant LT
  • a gene (gene 10) was prepared which kept the SD region and the initiation codon at a distance of 13 bases and simultaneously encoded the head of LT.
  • PtrpMLTDNA was digested with a restriction enzyme Nsil (NEB) — HindII (Toyobo), and then electrophoresed on a 1% low-melting point agarose gel to recover a gene consisting of 4655 bp (gene 8).
  • Nsil Nsil
  • HindII Toyobo
  • Bluescript M13S ⁇ (+) 2 / g (Toyobo) was digested with restriction enzyme BamHI-Hindll and then electrophoresed on a 0.8% low-melting-point agarose gel. After the r-staining, the desired plasmid DNA (gene 9) consisting of 2.9 kb was obtained.
  • T4 DNA ligase was allowed to act on genes 6, 8, 9, and 10 to obtain a plasmid (pB trp TLT) having the TLT gene. Further, the HB101 strain for transformation, which was frozen and stored at 170 ° C, was thawed, and 200 ng of pBTrp TLT was added to 100 ⁇ l of this suspension. did. After 4 5 minutes water cooled, 4 give 9 0 seconds heating preparative tio click at 2 e C, cooled for 2 minutes on ice.
  • TLT protein the peptide of the present invention
  • the obtained bacterial cell 16.92 gZwet (3.5 1 min) was transformed with 50 mM Tris-hydrochloric acid (pH 8.0) 120 containing 30 mM NaCl and O.lmMP-APMSF.
  • the suspension was then passed through a high-pressure homogenizer (RANNIE) three times at 600 psi to destroy the cells. Further, the inside of the high-pressure homogenizer was washed with 120 ml of the same buffer, and the recovered liquid and the disrupted liquid were centrifuged to obtain a crude extract.
  • RNIE high-pressure homogenizer
  • the crude extract 2 5 O ml to as the final concentration was added to port Riechiren'i Mi emissions to be 1% 0.
  • the eluate (flow-through fraction) 2501111 was adjusted to 117.0.
  • the resulting protein solution was adsorbed on a column of CM Sepharose Fast Flow (Pharmacia LKB), flattened with 5 mM phosphate buffer (PH 7.0), and 5 mM phosphate was added. After washing with a buffer solution (PH 7.0), elution was carried out with a stepwise concentration gradient using 5 mM phosphate buffer (pH 7.0) containing NaCl. Fractions containing the peptide were collected, dialyzed against a phosphate buffer, and the activity was determined by an L929 test. The specific activity and the recovery in each purification step were determined. The protein quantification was measured using a protein assay system manufactured by Piolado. Was. The results are shown in Table 3.
  • the peptide of the present invention was recovered with a purity of 95% or more.
  • FIG. 4 shows the nucleotide sequence and the peptide of the present invention which it encodes.
  • a T4 DNA ligase was allowed to act on genes 11 and 12, and a brassmid (p9trpHLT) having the MLT gene was obtained. Further, the HB101 strain cells for transformation which had been frozen and stored at ⁇ 70 ° C. were thawed, and 20 ng of p9 trp MLT was added to the suspension 1001. After 45 minutes the reaction water cooling, 4 2 e C with give 9 0 seconds human one preparative sucrose Uz click, SOC medium was added 9 0 cooled with 2 minutes ice, after 1 hour incubation at 37 hand, suspension 0 Transformed on a LB agar plate containing lmgZml of ampicillin and cultured overnight at 37 ° C to obtain a transformant having genes 11 and 12. DNA was isolated from this transformant, cut with the restriction enzyme EcoRI-Hindm, electrophoresed on a 1% agarose gel, and confirmed as a 629 bp fragment by EtBr staining. (Gene 11).
  • a single colony of P9 trp MLT was added to an M9CA medium containing 2.5 g ZmI tritophan (M9 minimal medium, 1% glucose, 1 mm Mg S O-, 0.1 m MC a C l s, 0.5% Kazami Roh acid, 0. 1 mg / ml Chia Mi down one HC 1, 0. 1 mg / in 1 Anne Pishi Li down, l OO m MH epes) Niu decorating, 3 7. C. overnight. The next day, it intends 1 5 0 amount to the new M 9 CA medium For example, OD B.
  • the remaining lml of the culture was separated by centrifugation.
  • the cells were suspended in lml of PBS (-) buffer solution, sonicated for 10 minutes, and then centrifuged. By separation, a protein supernatant was obtained. After the protein supernatant through a 0.2 2 Mi black down the full I le evening one, for comparison in the same L 9 2 9 cytotoxicity test, bacteria fluid lml those Ri 1 X 1 0 6 unit / ml of the active was confirmed.
  • Probe labeling was performed using these oligodeoxynucleotides at a final concentration of 1 O O pmo 1 l O O iaM Tris-HCl buffer (pH 8.0), 10 mM magnesium chloride, 5 mM DTT,
  • 5 unit T 4 port consists Li quinuclidine Leo Chi Dokinaze solution 3 0 added during 1, 3 7, allowed to react for 45 minutes and re-phosphorylated by, further, the enzyme To inactivate, the treatment was carried out at 65 ° C for 10 minutes.
  • the probe labeled according to the above (b) was individually associated with the filter to which the DNA of the above (a) was fixed. This association reaction was performed using a 6-fold concentration of SSC (0.15 M sodium chloride-0.015 M sodium citrate) containing a 277.5 KB q-labeled probe, and a 5-fold residue. Degree of den A solution consisting of a heart solution (Denhardt's), 0.1% SDS and 200 ⁇ g / ml denatured salmon sperm DNA. After immersion for 6 hours, after the reaction, the filter was immersed in 6 XSSC-0.1% SDS solution three times at 40 ° C for 15 minutes, washed, and further washed with 0.2 XSSC-0.1% SDS solution. Washed by immersion 3 times at 0 ° C.
  • Radioautograms were taken from the washed filters, and plaques that responded to the probe were searched for by overlaying a set of two Replicap filters with radioautograms. Ri by this method, Ri O 6 X 1 0 5 plaques to afford two EMBL 3 full Aji to respond to the probe.
  • the phage DNA was extracted and purified by a grise mouth-step gradient method [ManiattsT, Molecuulararcloning, p83 (1992)].
  • the obtained DNA was cleaved with restriction enzyme EcoRI (manufactured by Toyobo), separated by 0.5% agarose gel electrophoresis, and the DNA fragment was separated from the agarose gel by a Southern blot method using a BA852-torose cell mouth. Transcribed on a surface filter (S & S). The probe used in (1) was associated with this filter to obtain a reacted 2.4 kb DNA fragment. This DNA fragment completely contained the signal peptide portion of the human photophosphotoxin to the termination codon.
  • EcoRI restriction enzyme
  • S & S surface filter
  • DNA fragment recovered as described above is digested with the restriction enzyme Rsll (NEB), and a DNA fragment of 528 bp containing a part of exon 4 of the lymphotoxin gene (hereinafter referred to as “DNA fragment”).
  • the gene a) was isolated. This gene is shown below. 5 '-GACCGTGCCT TCCT CCAG
  • exon 3 and exon 4 of the lymphotoxin gene were synthesized using one or two oligodoxynucleotides (1) to (12) below, which were synthesized organically using a DNA synthesizer. And synthesized.
  • the mixture was subjected to 5% acrylamide gel electrophoresis. After staining with ethidium bromide, a 158 bp DNA fragment (hereinafter referred to as “gene b”) was cut out along with the gel and dissolved in an elution buffer. Elution was performed, and the Sephadex G25 gel filtration column (Bulmasia LKB) was desalted and concentrated.
  • gene b a 158 bp DNA fragment
  • This gene b is shown below.
  • Bluescript M1 3 KS (+) Restrict vector vector (Toyobo) After digestion with the enzyme EcoRI, the phosphoric acid at the 5 'end is removed by treatment with alkaline phosphatase, and the mixture of this and the gene a and gene b prepared as described above is added to the mixture. By reacting with T4 DNA ligase, plasmid 1 (blue script M13 KS (+) — gene a—gene b) having a structural gene for human lymphotoxin was obtained.
  • E. coli XL1 blue strain cells for transformation which had been frozen and stored at 170 ° C., were thawed, and 20 ng of plasmid 1 was added to the suspension 1001.
  • the gene a-b obtained in the above (3) was cut with a restriction enzyme PVuII (Toyobo) to obtain a DNA fragment of 6553 bp (hereinafter, referred to as gene c).
  • the gene c is shown below.
  • oligonucleotide was desalted and concentrated, dissolved in an annealing buffer, treated at 90 ° C. for 3 minutes, then slowly cooled and annealed to obtain gene d.
  • the gene d is shown below.
  • Blues script M13KS (+) vector (Toyobo) was cut with the restriction enzyme EcoRI, and the 5'-terminal phosphoric acid was removed by treatment with alkaline phosphatase. Then, this is mixed with gene c and gene d, and T4 DNA ligase is allowed to act thereon to produce plasmid 2 having gene c and gene d (Blues Ml 3 KS (+) — Gene c—gene d) was obtained.
  • transformant c-d a transformant having the gene c and the gene d (transformant c-d) was obtained.
  • the blue script M13KS (+) — gene c-gene d was isolated from the transformant c-d, cut with the restriction enzyme EcoRI, and then subjected to low melting. at point agar electrophoresis, structural gene 6 7 3 b P gene c and gene d are bonded (modified Li lymphotoxin butoxy down structural gene; gene c - d) were separated and recovered.
  • the expression vector pkk2 2 3 — 3 vector (Pharmacia LKB) was digested with the restriction enzyme EcoRI, and the 5′-terminal phosphoric acid was removed by treatment with alkaline phosphatase.
  • EcoRI restriction enzyme
  • alkaline phosphatase alkaline phosphatase
  • L 9 2 9 cells were suspended to be 2 X 1 0 5 cells / ml of ⁇ , which one 0 0 ⁇ 1 to 9 6-well plates dispensing Then, the cells were cultured at 37 ° C for 5 hours. Then, the peptide of the present invention 101 and serially diluted 10-fold and actinomycin D (2.5 g- / m 1) l O jl were added, and cultured at 37 for 18 hours. did.
  • the amount of biological activity required to kill 50% of L929 cells was defined as 1 unit, and the antitumor activity of the peptide of the present invention was determined. 9.48 was X 1 0 7 units / mg.
  • lymphotoxin recombinant lymphotoxin purified from the body of Escherichia coli and the peptide of the present invention at 4 ° C. was tested as follows.
  • the purity of the lymphotoxin and the peptide of the present invention in the electrophoresis immediately after the preparation was 95% or more for the lymphotoxin and 95% for the peptide of the present invention.
  • the lymphotoxin stored for months and that of the peptide of the present invention had a purity of 0% lymphotoxin and 60% of the peptide of the present invention.
  • Lymphotoxin is originally a protein with a molecular weight of 18,800, but analysis by swimming after storage for two months shows that it is a protein with a molecular weight of 17,000 and 16,000. Degraded, there was no lymphotoxin protein with a molecular weight of 18,800.
  • the peptide of the present invention is a protein having a molecular weight of 180,000, and after storage for 2 months, has a molecular weight of 17,000 and a molecular weight of 16,000, similar to lymphotoxin. A decomposition product of 0 was confirmed, but the peptide of the present invention having an original molecular weight of 18,000 Protein was also present at 60%.
  • the peptide of the present invention was excellent in storage stability as compared with that of linhotoxin.
  • An SD female rat (body weight: 197 to 21.3 g) was anesthetized by intraperitoneal administration of 5 ml of 25% urethane and fixed in the dorsal position. The experiment was performed after about 10 minutes with the jugular vein visible. Administration of each protein and blood collection were performed from the right jugular vein. The dose of each protein was 250 g rat. Immediately after administration to the rat (0 minute), 5, 10, 15, 20, 40, 60, and 90 minutes later, approximately 0.15 ml of blood is collected, and the blood is promptly collected at each blood collection.
  • both the recombinant lymphotoxin and the peptide of the present invention were 12 minutes, and the peptide of the present invention could maintain almost the same blood activity as the lymphotoxin. confirmed.
  • the present invention paves the way for the industrially advantageous production of LT and its clinical use, and is extremely useful.

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Abstract

L'invention se rapporte à un vecteur avec lequel on peut exprimer efficacement des lymphotoxines, y compris la lymphotoxine naturelle et des peptides du type lymphotoxine ayant une forte homologie avec la lymphotoxine; ainsi qu'à un procédé pour produire des lymphotoxines au moyen d'un tel vecteur. Ce vecteur comporte la séquence SD située 13 bases en amont du codon initiateur, une séquence de base de TTAACT dans la région -10 et une séquence de base de TTGACA dans la région -35, ainsi qu'un poids moléculaire de 2,7 à 2,1 kb, à l'exception du gène codant pour les lymphotoxines. Ces lymphotoxines sont produites essentiellement par recombinaison génétique et l'utilisation dudit vecteur sert à accroître considérablement la quantité d'expression de ces lymphotoxines, de sorte qu'on peut en faire un usage clinique.
PCT/JP1992/000043 1992-01-21 1992-01-21 Lymphotoxines, vecteur d'expression pour lymphotoxines, et production de lymphotoxines au moyen de ce vecteur Ceased WO1993014203A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0672681A4 (fr) * 1993-10-01 1997-02-26 Akira Kaji Nouveau peptide antitumoral.
US6287759B1 (en) 1992-09-18 2001-09-11 The United States Of America As Represented By The Department Of Health And Human Services Recombinant proteins of a Pakistani strain of hepatitis E and their use in diagnostic methods and vaccines

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JPS6211095A (ja) * 1985-07-04 1987-01-20 Kanegafuchi Chem Ind Co Ltd リンホトキシン
JPH02447A (ja) * 1987-10-27 1990-01-05 Sankyo Co Ltd ヒトリンホトキシン合成遺伝子
JPH0352822A (ja) * 1989-07-21 1991-03-07 Denki Kagaku Kogyo Kk 複合制癌剤
JPH03106821A (ja) * 1989-09-20 1991-05-07 Denki Kagaku Kogyo Kk 抗腫瘍剤

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JPS6211095A (ja) * 1985-07-04 1987-01-20 Kanegafuchi Chem Ind Co Ltd リンホトキシン
JPH02447A (ja) * 1987-10-27 1990-01-05 Sankyo Co Ltd ヒトリンホトキシン合成遺伝子
JPH0352822A (ja) * 1989-07-21 1991-03-07 Denki Kagaku Kogyo Kk 複合制癌剤
JPH03106821A (ja) * 1989-09-20 1991-05-07 Denki Kagaku Kogyo Kk 抗腫瘍剤

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6287759B1 (en) 1992-09-18 2001-09-11 The United States Of America As Represented By The Department Of Health And Human Services Recombinant proteins of a Pakistani strain of hepatitis E and their use in diagnostic methods and vaccines
US6696242B1 (en) 1992-09-18 2004-02-24 The United States Of America As Represented By The Department Of Health And Human Services Recombinant proteins of a Pakistani strain of hepatitis E and their use in diagnostic methods and vaccines
EP0672681A4 (fr) * 1993-10-01 1997-02-26 Akira Kaji Nouveau peptide antitumoral.

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