RU2712519C1 - Recombinant plasmid expressing a cloned vibrio cholerae neuraminidase gene, and an escherichia coli strain - a superproducer of vibrio cholerae neuraminidase - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Disclosed is a recombinant plasmid pNanH expressing a cloned gene nanH (neuraminidase) Vibrio cholerae, built-in by BamHI-PstI sites in polylinker vector plasmid pQE30, under control of T5-promoter. What is also presented is an E.coli strain – a neuraminidase NanH Vibrio cholerae superconcentrate produced by transformation of the E.coli JM103 strain with the above recombinant plasmid. Invention can be used for preparing the V.cholerae neuraminidase preparations for the purpose of creating specific diagnosticums and pharmaceutical preparations, as well as for studying the properties, biochemical and biological activity of V.cholerae neuraminidase (NanH).

EFFECT: advantages of produced producer is high yield of desired protein and absence of ability to synthesis of any additional biologically active substances, which complicate its isolation and purification, possibility of cultivation without observing the mode of operation with agents of especially dangerous infections, accelerated preparation of neuraminidase.

2 cl, 3 dwg, 3 ex

Description

The invention relates to biotechnology, genetic engineering, medical microbiology, and can be used to obtain preparations of the neuraminidase (NanH) of Vibrio cholerae in order to create specific diagnosticums and pharmaceutical preparations, as well as to study the properties, biochemical and biological activity of the neuraminidase (NanH) of Vibrio cholerae.

Neuraminidase (NanH) is one of the important factors of the pathogenicity / persistence of cholera vibrios. On the one hand, it increases the sensitivity of intestinal cells to the action of cholera toxin, on the other hand, they participate in the utilization of higher-order gangliosides as a source of nutrition. It is known that NanH products can play a role in the development of a mild form of infection or short-term carriage [1-3]. This determines the relevance of further research on the properties of this factor, and requires the availability of its preparations.

NanH preparations are also widely used in biochemical and medical research, in the pharmaceutical industry [4]. The prospects of their use for the creation of new oral agents for the treatment of allergic diseases has been shown [5].

Commercial preparations of Vibrio cholerae neuraminidase produced by foreign companies (Sigma-Aldrich, ChemNet) are notable for their high cost (about 17,000 euros per 0.2 μM product). To produce them, toxigenic strains of cholera vibrios are used, which requires compliance with the regime of work with pathogens of especially dangerous infections. According to Taylor et al. [6], the product yield is about 1 mg / l of culture fluid. Bulgarian researchers proposed a more efficient producer as a producer — the non-toxigenic strain V. cholerae nonO1 / nonO139V13 [4], promising for industrial use, however, even in this case, the yield of purified protein is ~ 2 mg / l of culture fluid.

The most effective way to obtain this factor in preparative amounts is the use of laboratory E. coli strains containing and expressing the cloned nanH gene.

For the prototype, the previously designed Vimr E.K. et al. [7] E. coli strain HB101 pCVD364 containing 4.8 kb DNA fragment in the recombinant plasmid pCVD364 with the nanH gene expressed under the control of its own promoter. The activity of the product in extracts of cells carrying this plasmid strain E. coli HB101 amounted to 858 units / ml of total protein. The authors managed to isolate the purified preparation from these extracts, however, information on the amount of biomass used and the product yield was not given in the publication.

The disadvantage of the prototype is the uncontrolled expression of the NanH gene and a low yield of the desired protein, for the isolation of which in preparative quantities, it will be necessary to build up producers in large volumes.

The technical task of the invention is the cloning of the nanH gene in the plasmid vector pQE30, which provides the expression of foreign genes under the control of the powerful T5 promoter, and the creation of E. coli strain - super producer of recombinant V. cholerae protein NanH to isolate the desired product in preparative quantities from the minimum volumes of biomass.

The problem is solved by creating:

- a new recombinant plasmid pNanH expressing the cloned nanH gene of cholera vibrio in E. coli strains.

a strain of Escherichia coli JM103 pNanH, a superproducer of cholera vibrios neuraminidase by transforming the E. coli strain JM103 with the recombinant plasmid pNanH.

The pQE30 vector plasmid carries the ampicillin resistance gene (bla) and contains the promoter operator region, including the T5 promoter and two tandem lac operators, which maximize repression of NanH synthesis in the presence of glucose, as well as a synthetic ribosomal RNA binding site, start codon , a sequence of triplets encoding the synthesis of hexahistidine (6-His), polylinker (MCS) and two transcription terminators (t0 phage lambda and T1 from the rrnB operon of E. coli). Expression of cloned genes - occurs by induction with isopropyl-β-D-thiogalactoside (IPTG) and begins with a plasmid start codon, and a hybrid protein is formed with the hexahistidine block in front of the first amino acid.

Plasmid pNanH is a genetic engineering variant obtained by inserting the V.Holerae O1 nanH gene biovarEl Tor into the pQE30 vector (see Fig. 1).

Being transformed into a strain of E. coli JM103, a recombinant plasmid expresses the cloned gene under the control of the T5 promoter. Gene expression is suppressed in the presence of glucose and IPTG is induced.

The E. coli strain JM103 pNanH is a genetically engineered variant obtained by transformation of the recombinant plasmid pNanH into E. coli strain JM103 and is a producer of V. cholerae NanH. The strain is deposited in the State collection of pathogenic bacteria "Microbe" under the number KM2047.

The resulting producer strain is characterized by the following features:

Cultural and morphological properties

In liquid nutrient media (Hottinger broth, meat-peptone broth) forms a uniform turbidity, on dense - round, convex, smooth, white translucent colonies with a smooth edge, a doughy consistency.

Physiological and biochemical properties

The strain decomposes glucose, arabinosui mannitol with the formation of acid and gas, does not decompose sucrose, and forms lactose-negative colonies on the Endo medium. Auxotroph.

Neuraminidase activity: Glow in UV light after 20 minutes incubation for 20 μl of the cell culture of the strain with an equal volume of 4-methylumbelliferyl-N-acetylneuraminic acid substrate (1 mg / ml)

Antibiotic resistance

The strain is resistant to 50-100 μg / ml ampicillin due to the expression of the bla gene, which is part of the vector plasmid pQE30.

The method of obtaining and using a recombinant plasmid and producer strain is illustrated by the following examples.

Example 1. Cloning of the nanH gene and obtaining a recombinant plasmid.

The design scheme for the recombinant plasmid pNanH is shown in FIG. 1.

For PCR synthesis of the nanH gene, primers are used designed by the applicants based on an analysis of the nucleotide sequence of the VC1784 gene as part of the V.cholerae N16961 large chromosome:

straight -

the reverse is

Since amplitudes need to be inserted into the pQE30 plasmid vector in an orientation that ensures the direction of transcription under the control of the T5 promoter, a restriction site for the endonuclease forming sticky ends is inserted at the 5'-end of each primer: BamHI for the forward primer and PstI for the opposite (in the above sequences) shown in bold and underlined) in accordance with the order of the restriction sites in the polylinker of the vector plasmid.

From the V. cholerae strain El TorR-5879 Inaba (Museum of Living Cultures of the Rostov-on-Don Antiplague Institute), the nucleotide sequence of which nanH gene (KU215667) is completely identical to that of the reference strain V. cholerae N16961, chromosomal DNA is isolated by the phenolic method, which serves matrix for the synthesis of the desired gene.

300 μl of the reaction mixture for the polymerase chain reaction contain 0.5 ng of the DNA template and the following components in the indicated concentrations: 2.5 μl of each primer, 2.5 mm of all four deoxynucleotide triphosphates, 3 units Taq polymerase and 0.1 volume of 10-fold buffer attached to it. The mixture is poured into 30 μl into 0.5-ml plastic tubes and the reaction is carried out according to the following scheme: 94 ° С - denaturation (40 sec), 60 ° С - annealing (40 sec), 72 ° С - synthesis (40 sec). A total of 30 cycles of amplification are carried out, in the last cycle the synthesis time is increased to 3 minutes. At the end of the reaction, the contents of the tubes are combined, purified with a mixture of phenol: chloroform: isoamyl alcohol in a ratio of 25: 24: 1 and precipitated with ethyl alcohol.

The 2484 bp PCR amplification obtained in this way and the DNA of the vector plasmid pQE30 is hydrolyzed with restriction endonucleases BamHI and PstI according to the recommendations of the manufacturer of the enzymes, purified with a mixture of phenol: chloroform: isoamyl alcohol (25: 24: 1) and precipitated with ethyl alcohol. The precipitate is dissolved in a minimal volume of deionized water or is ligated using T4 phage DNA ligase and the buffer attached to it according to the manufacturer's recommendations.

Ligase mixtures transform competent E. coli Jm103 cells prepared the day before by treatment with calcium chloride. After the standard transformation procedure (0 ° C - 40 min, 42 ° C - 2 min, 0 ° C - 5 min), the cells are diluted 10 times with LB medium with 0.5% glucose, grown for 1 h and plated on LB agar containing 50 μg / ml ampicillin and 0.5% glucose. Crops are incubated at 37 ° C. On the next day, recombinant clones were selected by PCR with primers to detect the desired nanH clone:

straight -

the reverse is

The DNA of the donor strain P-5879 serves as a positive control, and the E. coli strain Jm103pQE30 containing a vector plasmid without insertion as a negative control. The results are taken into account after electrophoresis of the reaction mixtures in a 1.8% agarose gel by the presence / absence of an amplification of 585 bp in length.

Example 2. The study of the expression of the cloned nanH gene in E. coli

To test the ability of recombinants to express the gene nan, the clones selected in Example 1 were cultured in LB broth containing 50 mg / ml ampicillin (without glucose) at 37 ° C for 3-4 h followed by the induction of IPTG at a final concentration of 1 mM for 1 h. Then, 20 μl of each culture is mixed on glass with an equal volume of a solution of 4-methylumbelliferyl-N-acetylneuraminic acid substrate (1 mg / ml), after 20 minutes they are viewed under UV light and fluorescence is observed in samples of recombinants capable of NanH synthesis. In the control E. coli strain Jm103pQE30E it is absent (Fig. 2). The positive clone with the best glow is selected for further research.

In FIG. 2. The production of NanH by recombinant E. coli Jm103pNanH clones is shown: luminescence in UV light after incubation with the substrate. 1 - control strain of E. coli Jm103 containing pQE30 vector plasmid without insertion, 6 - NanH-negative clone, 2-5, 7 - NanH-positive clones, 8 - V. cholerae P-5879 culture.

Example 3. The study of the productivity of the strain E. coli Jm103pNanH and localization of the recombinant protein in its cells.

The recombinant E. coli strain JM103pNanH (KM 2047), as well as the control strain containing the vector plasmid pQE30 without insert, are grown in LB liquid medium containing 50 μg / ml ampicillin (without glucose) for 3-4 hours at 37 ° C shunting at 150 rpm and then inducing 1 mM IPTG for 1-2 hours, cells are pelleted by centrifugation, lysed in buffer containing 65 mM Tris-HClPH 6.8, 1% SDS and 10 mM 2-mercaptoethanol, at a temperature 99 ° C for 10 minutes The lysate was subjected to electrophoresis on a 10% polyacrylamide gel (SDS page) with SDS and Coomassi Blue R250 gel was stained. Major protein bands were detected in the E. coli JM103pNanH cell lysate in the region with MM of ~ 83 kDa and ~ 89.5 kDa (which corresponds to the molecular weight of the two forms of NanH), in contrast to the lysate of the control strain containing a vector plasmid without insertion (Fig. 3A - lysates of whole cells). According to the Quantity One program, their total specific gravity is approximately 9-10% of the total cellular proteins.

To determine the localization of the recombinant protein, cells of the JM103pNanH strain grown by induction are destroyed by ultrasound on a QSonica Q700 disintegrator for 10 min (40 pulses of 5 sec, 357 J with interruptions of 10 sec; amplitude of 50) and subjected to electrophoresis of soluble (os) and insoluble (n / a) fraction of cells separated by centrifugation (Fig. 3B).

In FIG. 3B displays: _ insoluble (os) and soluble (n / a) fractions of ultrasonic disintegrators Jm103 p NanH.

The form with a molecular weight of ~ 89.5 kDa is an unprocessed protein with a hexatidine block (6His-tag) at the N-terminus and is located in the insoluble fraction of cells in the form of inclusion bodies. Its specific gravity is 5.6-6.6% of total cellular proteins. The second is found mainly in the soluble fraction, i.e. in the periplasmic space of cells. Its molecular weight of ~ 83 kDa corresponds to the mature form of NanH, which is formed due to the removal of the signal sequence together with 6His tag, with a total length of 58a. The specific gravity of the mature form is 3.4-3.8% of the total proteins.

Thus, the E. coli strain M103pNanH-superproducer of V. cholerae El Tor neuraminidase was constructed, which can be used to isolate the target product in preparative amounts. Depending on the tasks of further research, it is possible to obtain the starting product (6His-NanH) from inclusion bodies using a specific sorbent of the Ni-NTA type (nickel-nitrile acetylated sepharose), or a mature NanH protein from clarified ultrasonic disintegrates of cells using other purification methods. The advantages of the obtained producer in comparison with cholera vibrios are a high yield of the desired protein, the possibility of cultivation without observing the regime of work with causative agents of especially dangerous infections, the lack of the ability to synthesize any additional biologically active substances that could complicate its isolation and purification, and compared with known recombinant producer strains - a short period of biomass growth (4-6 hours including induction), will provide accelerated preparation ata.

Sources of information

1. Galen J.E., Ketley J.M., Fasano A., Richardson S.N., Wasserman S.S., Kaper J.B. Role of Vibrio cholerae neuraminidase in the function of cholerae toxin. Infect. Immun. 1992; 60 (2): 406-415.

2. Jermyn W.S., Boyd E.F. Characterization of a novel Vibrio pathogenicity island (VP1-2) encoding neuraminidase (nanH) among toxigenic Vibrio cholerae isolates. Microbiology. 2002; 148: 3681-3693.

3. Figueiredo S.C., Neves-Borges A.C., Coelho A. The neuraminidase gene is present in the non-toxigenic Vibrio cholerae Amazonia strain: a different allele in comparison to the pandemic strains. Mem. Inst. Oswaldo Cruz. 2005; 100 (6): 563-569.

4. Eneva R.T., Engibarov S.A., Petrova P., Abrashev R., Strateva T., Kolyovska V., Abrashev J. High production of neuraminidase by a Vibrio cholerae non-01 strain - the first possible alternative to toxigenic producers. Appl. Biochem. Biotechnol. 2015; 176 (2): 412-427.

5. Diesner SC, Bergmayr C., Wang XY, Heiden D., Exenberger S., Roth-Walter F., Starkl P., Ret D., Palischoll J., Gabor F., Untersmayr E. Characterization of Vibrio cholerae neuraminidase as an immunomodulator for novel formulation of oral allergy immunotherapy. Clin. Immunol. 2018; 192: 30-39.

6. Taylor G., Vimr E., Garman E., Laver G. Purification, crystallization and preliminary crystalografic study of neuraminidase from Vibrio cholerae and Salmonella typhimurium LT2. J. Mol. Biol. 1992; 226 (4): 1287-1290.

7. Vimr E.R., Lawrisuk L., Galen J., Kaper J.B. Cloning and expression of the Vibrio cholerae-neuraminidase gene nanH in Escherichia coli. J. Bacteriol. 1988; 170: 1495-1504.

Claims (2)

1. Recombinant plasmid pNanH expressing the cloned nanH (neuraminidase) Vibrio cholerae gene inserted at the BamHI-PstI sites in the polylinker of the vector plasmid pQE30 under the control of the T5 promoter. 2. The strain Escherichia coli KM 2047 State collection of pathogenic bacteria "Microbe", carrying the recombinant plasmid pNanH according to claim 1 - superproducer of neuraminidase NanH Vibrio cholerae.

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