KR19980075704A - Recombinant microorganism expressing the antigenic protein CagA of Helicobacter pylori - Google Patents

Abstract

The present invention is a fusion in which the A2 / B subunit gene is linked to the antigenic protein CagA (CagA) gene of Helicobacter pylori and the toxin gene of Vibrio cholerae. Method for preparing chimeric protein in which CagA of Helicobacter pylori and A2 / B subunit of Vibrio correia toxin are fused using an expression vector containing a gene, a recombinant microorganism transformed therewith, and an electric expression vector. It is about. The recombinant DNA is artificially designed to facilitate its expression and genetic manipulation, and also exhibits excellent immunogenicity against Helicobacter pylori, stable to the environment within the stomach, and easily penetrates the gastric mucosa to secrete immunoglobulin (sIgA). It is made up of a combination of genes that provide proteins that can accelerate the production of maximal industrial utility. Therefore, the chimeric protein expressed from such recombinant DNA can be used as an active ingredient in the production of a diagnostic reagent for Helicobacter pylori or a vaccine for the prevention or treatment of Helicobacter pylori related diseases, It can also be used to generate antibodies against.

Description

Recombinant Microorganism Expressing Helicobacter Pylori Antigen Protein CagA

The present invention relates to recombinant microorganisms transformed with recombinant DNA expressing the antigenic protein CagA (cytotoxin-associated gene A) of Helicobacter pylori and electro recombinant DNA. More specifically, the present invention provides a fusion gene in which the A2 / B subunit gene is linked to the CagA gene encoding the epitope of Helicobacter pylori and the toxin gene of Vibrio cholerae. A method for producing a chimeric protein in which CagA of Helicobacter pylori and A2 / B subunits of Vibrio correrai toxin are fused using an expression vector comprising the recombinant vector transformed with the recombinant microorganism and the electric expression vector. will be.

Gastritis-related diseases such as gastritis and gastroduodenal ulcers can be caused by a variety of causes, but are mainly caused by Helicobacter pylori inhabiting the epithelial junctions of the gastric mucosa (Marshall, BJ, J. Infect. Dis., 153: 650-657 (1986); Maaroos, HI et al., Scand. J. Gastroenterol., 26 (suppl. 186): 65-72 (1991); Walsh, JH and Peterson, WI, N. Engl. J. Med., 333 (15): 984-991 (1995); Tytgat, GNJ, Scand. J. Gastroenterol., 31 (suppl.215): 70-73 (1996)). Although there are regional differences, more than 90% of Asians and 60% of Europeans are infected with H. pylori (Ateshkali A. et al., Clin. Pharm., 12: 34-48 (1993) Tompkins LS and Falkow S., Science, 267: 1621-1622 (1995); Sanders SW, Clin. Ther., 18 (1): 2-34 (1996)), and also gastritis and gastroduodenal ulcers Relapse by Helicobacter pylori, drug-resistant even after treatment with agents (Coghlan, JG et al. Lancet, 2: 1109-1111 (1987); Tatsuta, M. et al., Gut , 31: 973-976 (1990); Graham, DY et al., Ann.Intern.Med., 116: 705-708 (1992)), eventually known to progress to gastric cancer after many years (Tsujii, M. et al., Cancer Lett., 65: 15-18 (1992); Parsonnet, J. et al., N. Engl. J. Med., 325: 1127-1131 (1991); The Eurogfgast Study Group. Lancet, 341: 1359-1362 (1993); Forman, D. et al., Brit. Med. J., 302: 1302-1305 (1991).

In order to treat gastritis-related diseases caused by Helicobacter pylori, various chemotherapeutic agents, including antibiotics or anti-ulcers, have been used. However, these drugs have limited permeation of gastric mucosa. There was a problem causing a significant increase in resistant bacteria, recurrence of infection, side effects of drugs, and the like.

Therefore, there is an urgent need for the development of a novel drug that can remove the Helicobacter pylori bacteria by immunotherapy rather than chemotherapy. Therefore, in recent years, in order to solve the above problems, the development of a vaccine (vaccine) against Helicobacter pylori bacteria has been attempted. That is, CagA (cytotoxin-associated gene A), which is a gene encoding the epitope of Helicobacter pylori, which has been identified so far (Covaccin, A. et al., Proc. Natl. Acad. Sci., USA, 90: 5791 -5795 (1993)), urease (Labigne, A. et al., J. Bacteriol., 173: 1920-1931 (1991)), flagella (Leying, H. et. al., Mol. Microb., 6 (19): 2863-2874 (1992)), adhesin (Evans, PG et al., Gene, 163: 97-102 (1995)), superoxide Superoxide dismutase (Spiegelhalder, C. et al., Infect. Immun., 61: 5315-5325 (1993)), catalase (Hazell, SL et al., J. Gen) Microbiol., 137: 57-61 (1991)), vacuolating cytotoxin (Telford, JL et al., J. Exp. Med., 179: 1653-1658 (1994)) genes, etc. Vaccines for the diagnosis and prevention of Helicobacter pylori strains have been developed, but some vaccines are in the preclinical stage, but have various disadvantages in commercialization. It has not yet been commercialized up to the door. For example, vaccines using urease genes are poorly immunogenic; Vaccines using vacuogenic cytotoxin genes are highly immunogenic, but the toxicity of cytotoxin itself may be a problem; Vaccines using the adhesin gene are excellent in immunogenicity, but do not promote the production of secretory immunoglobulins (secretory IgA: sIgA), thereby reducing their efficacy as vaccines. In addition, the vaccine using the CagA gene has a disadvantage in that the gene is not present in all Helicobacter pylori bacteria because it is not present in all Helicobacter pylori bacteria, but is found in gastric cancer patients. Cobacter pylori has a CagA gene which can be effectively used for the prevention and treatment of gastric cancer.

In addition, because Helicobacter pylori bacteria inhabit the epithelial junctions of the gastric mucosa, the bacteria are removed by secretory immunoglobulins rather than humoral immunoglobulins, and such vaccines do not easily penetrate the gastric mucosa. Due to its inability to stimulate the mucosal immune system, its ability to promote the production of secretory immunoglobulins (sIgA) is reduced, thus reducing the immune effect against Helicobacter pylori and also gastric acid (pH 1). It had a serious problem that it denatures easily by -2), and activity falls.

Since the vaccine developed using the Helicobacter pylori gene alone has various problems described above, the present inventors easily penetrate the gastric mucosa and stimulate the mucous immune system to generate secretory immunoglobulin (sIgA). A protein having a function of inducing a chimeric protein combined with a fusion partner was prepared and used as a vaccine.

First, the present inventors paid attention to the following features of the Vibrio correia toxin gene during the search for a fusion partner exhibiting the above functions. The Vibrio Correia toxin gene consists of three subunits, A1, A2, and B (Mekalanos, JJ et al., Nature, 306: 551-557 (1983)). Toxic, the A2 subunit gene connects five B subunits into one molecule, and the B subunit binds to the host cell to promote sIgA production and enhance the stability of the protein to the surrounding environment. Part. In addition, while various vaccines using the entire intact cholera toxin as a fusion partner cannot be directly used in humans due to the toxicity of the A1 subunit, the vaccine was prepared using the A2 / B subunit of the Vibrio cholera toxin gene. Vaccines can be used in humans and are very useful. Thus, as a result of the study of the vaccine prepared using the Vibrio Correra A2 / B subunit as a fusion partner, chimera by binding the Adhesin gene of Streptococcus mutans to the Vibrio Correra A2 / B subunit Producing proteins and administering them in oral vaccines has been reported to not only promote the production of secretory immunoglobulins (sIgA) and humoral immunoglobulins (serum IgG), but also continue to produce antibodies for more than six months. (Hajishengallis, G., J. Immunol., 154: 4322-4332 (1995)).

Against this background, the inventors of the present invention have successfully synthesized chimeric proteins from recombinant expression vectors comprising a fusion DNA linking the CagA gene in the Helicobacter pylori epitope coding gene and the A2 / B subunit gene in the Vibrio chorerai toxin gene. Expression was found and the present invention was completed. Thus expressed chimeric protein exhibits excellent immunogenicity against Helicobacter pylori, stable to the environment within the stomach, and easily penetrates the gastric mucosa to promote the production of secretory immunoglobulins (sIgA), thereby preventing the problems of the conventional vaccines described above. The solution can be used as an effective vaccine against Helicobacter pylori.

After all, the first object of the present invention is to provide a nucleotide sequence of the fusion DNA connecting the CagA gene of Helicobacter pylori and the A2 / B subunit gene in the Toxin gene of Vibrio correrai and the amino acid sequence translated therefrom. .

It is a second object of the present invention to provide an expression vector comprising the fusion DNA sequence and a microorganism transformed therewith.

A third object of the present invention is to provide a method for producing a chimeric protein in which the antigenic protein CagA of Helicobacter pylori and the A2 / B subunit of Vibrio correia toxin are fused from an electrotransformed microorganism.

Figure 1 shows the nucleotide sequence of the fusion gene connecting the CagA gene of Helicobacter pylori and the A2 / B subunit gene of Vibrio correia toxin.

Figure 2 shows the amino acid sequence translated from the nucleotide sequence of the fusion gene described in Figure 1.

3 is a schematic diagram showing the construction of the Helicobacter pylori CagA expression vector pHC033.

Figure 4 is a photograph showing the result of 12% SDS-PAGE of the cell lysate of E. coli transformed with the expression vector pHC033.

Hereinafter, the present invention will be described in more detail.

In order to promote antibody production against Helicobacter pylori, chimeric proteins were prepared using the gene of Helicobacter pylori CagA having excellent immunogenicity. That is, the A2 / B subunit genes of the Helicobacter pylori CagA gene and the Vibrio correia toxin gene were respectively prepared by polymerase chain reaction (PCR), and then each fragment gene was digested with restriction enzyme EcoRⅠ. The two genes were combined with T ₄ DNA ligase. The fusion genes thus linked were cleaved with an appropriate restriction enzyme and linked to a plasmid having a promoter for expressing the protein to prepare a recombinant expression vector. Preferably, the fusion gene was digested with restriction enzymes DsaI and PstI and inserted into the pTED plasmid to prepare expression vector pHC033. E. coli was transformed with the expression vector and cultured of recombinant E. coli, and then chimeric proteins in which CagA of Helicobacter pylori and A2 / B subunits of Vibrio correrai toxin were fused were obtained.

In the amino acid sequence of the present invention, among functionally equivalent Iran amino acid sequences of chimeric proteins, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; And all proteins substituted with a combination such as Phe and Tyr. In addition, in the nucleotide sequence of the present invention, the functional equivalent means all genes having a nucleotide sequence capable of providing an amino acid of the electric combination in the nucleotide sequence of the fusion gene.

The chimeric protein prepared above can be used as an active ingredient in the preparation of a diagnostic reagent for Helicobacter pylori or a vaccine for the prevention or treatment of Helicobacter pylori-related diseases, and also for the production of antibodies to Helicobacter pylori. Can be.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Isolation of Chromosome DNA of Helicobacter Pylori

Helicobacter pylori 11637 RPH 13487 (ATCC 43504) was added to 5% of serum serum infusion of BHI (brain heart infusion) (vancomycin) 10mg / ml, trimetofrim 5mg / ml, amphotericin Ratio (amphotericin B) consisting of 4 mg / ml) was incubated for 72 hours in a 10% CO ₂ incubator, and then chromosomal DNA was isolated in a conventional manner.

Example 2: Synthesis, Purification and Analysis of Oligonucleotides

Two oligonucleotides, 37 and 30, were used to synthesize CagA gene amplification of Helicobacter pylori (Example 3 below):

5'-CCGTGGATGACTAACGAAACCATTGACCAACAACCAC-3 '

5'-AGAATTCTTAAGATTTTTGGAAACCACCTT-3 '

In addition, two oligonucleotides, 28 and 27 mer, were used to amplify the A2 / B subunit gene amplification of the Vibrio chorerai toxin gene (Example 3 below):

5'-AGAATTCGAAGAGCCGTGGATTCATCAT-3 '

5'-ACTGCAGCACATAATACGCACTAAGGA-3 '

In the above, oligonucleotides were synthesized using an automated nucleotide synthesizer (Pharmacia LKB Biotechnology, Uppsala, Sweden).

Each oligonucleotide thus synthesized was reacted with a solution of TTD (thiophenol / triethylamine / dioxane = 1/2/2 (v / v / v)) attached to silica, washed well with methanol and ethanol, and After treatment with strong ammonia water, the synthetic oligonucleotides were separated from the silica matrix. Strong ammonia water was further added to the oligonucleotide solution thus separated and reacted at 50 ° C. for 12 hours, and the solution was concentrated under reduced pressure until the solution became 0.5 ml with gas removal. The concentrated oligonucleotides were then first purified with acetonitrile / triethylamine buffer using a SEP-PAK cartridge (Waters Inc., USA) and 15% polyacrylamide gel (TE-boric acid, pH 8.3). Each was electrophoresed. After electrophoresis, the location of the oligonucleotides was confirmed by short wavelength ultraviolet rays, and only the site was cut. Oligonucleotides were then extracted from the cut gel sections, and salts were removed with acetonitrile / triethylamine buffer using a SEP-PAK cartridge connected to a syringe to purify each oligonucleotide. The purified oligonucleotides of each nucleotide is T ₄ polynucleotide kinase (New England Biolabs, # 201S, USA) r- ^"32 P" labeled -ATP and maksam Gilbert sequencing using (see: Maxam, AM Gilbert, W , Proc. Natl. Acad. Sci., USA, 74: 560-564 (1977)) to determine the sequence of each oligonucleotide.

Example 3 Polymerase Chain Reaction (PCR)

Template DNA (10 ng to 100 ng), 10 μl of 10 × Taq polymerase buffer (500 mM KCl, 15 mM MgCl ₂ and 10 mM Tris-HCl with 0.1% (v / v) gelatin, pH 8.3), 10 μl of dNTP's mixture ( 1 g of dGTP, dATP, dTTP, and dCTP, each containing 1.25 mM), 2 μg of primer (oligonucleotide synthesized in Example 2), and 0.5 μl of Ampli Taq DNA polymerase (Perkin Elmer Cetus, USA) in distilled water Was added to bring the total volume to 100 μl. To this was added 50 μl of mineral oil to prevent evaporation of the solution. At this time, in the case of amplifying the CagA gene of Helicobacter pylori, the template DNA is the chromosomal DNA of Helicobacter pylori isolated in Example 1, and the primer is a 37mer and 30mer oligo synthesized in Example 2 Nucleotides; In the case of amplifying the A2 / B subunit gene of the Vibrio correia toxin gene, the template DNA is the chromosomal DNA of the Vibrio correra, and the primers are 28 and 27 mer oligonucleotides synthesized in Example 2.

For PCR reactions, denaturation (95 ° C, 1 minute), annealing (55 ° C, 1 minute), extension using a temperature cycler (Thermal Cycler, Cetus / Perkin-Elmer, USA) (72 ° C., 2 minutes) was performed 30 times, and finally, further reacted at 72 ° C. for 10 minutes. Then, to remove the polymerase, an equal volume of phenol / chloroform mixed solution (1: 1 (v / v)) was added to the reaction mixture, and the mixture was mixed well and centrifuged. The supernatant was transferred to a new tube, 1/10 volume of 3M sodium acetate, 2 volumes of 100% ethanol were added and mixed, followed by centrifugation to obtain a double helix nucleic acid. This nucleic acid was dissolved in 20 μl of TE buffer and used for later experiments.

Example 4 Preparation of Expression Vector pHC033

For the fusion of the genes, the A2 / B subunit genes of the Helicobacter pylori CagA gene and the Vibrio correrai toxin gene amplified in Example 3 were respectively digested with EcoRⅠ restriction enzymes, and then 1 μg of DNA was mixed and mixed 10 times. Adjust the total reaction volume to 30 μl by adding 3 μl of conjugated concentrate (600 mM Tris-HCl, pH 7.5) containing 10 mM DTT and 100 mM MgCl ₂ , ₁ μl of 10 mM ATP and 10 units of T ₄ DNA ligase. And reacted at 14 ° C. for 16 hours. The reaction was subjected to 1% agarose gel electrophoresis to obtain a fusion gene on the order of 4.1 kb and its nucleotide sequence was determined (see FIG. 1). In FIG. 1, bases 1 to 3444 represent the sequence corresponding to the structural gene of CagA, and bases 3445 to 3477 represent the sequence corresponding to the signal peptide of the Vibrio Correlai B subunit. Figure 2 shows the amino acid sequence translated from the base sequence of the fusion DNA described in Figure 1.

The fusion gene having the nucleotide sequence determined above was double-cut with restriction enzymes DsaI and PstI, and then inserted into the gene carrier pTED, double-cut with electric restriction enzyme, to prepare a cyclic plasmid, which was named pHC033. The plasmid pTED is a 2.95 kb plasmid that generates a DsaI restriction enzyme recognition site in pTE105 isolated from Escherichia coli JM101 (DW / BT-2042) (KCCM 10027), a transformant of pTE105 (see Figure 3). ). 3 is a diagram showing the construction of the expression vector pHC033.

In addition, the restriction vector was treated in the expression vector pHC033, and 1% agarose gel electrophoresis confirmed that a single restriction enzyme site was present in the expression vector pHC033. Thus, the Helicobacter pylori CagA gene and Vibrio correra A2 / B It was found that the subunit gene was correctly inserted.

Example 5 Preparation of Transformant

In order to introduce the expression vector pHC033 into the host cell, E. coli JM101 strain was first inoculated into a liquid LB medium, incubated at 37 ° C. until absorbance at 650 nm reached 0.25 to 0.5, and then the cells. Was obtained and washed by adding 0.1M MgCl ₂ . Subsequently, the precipitate was obtained by centrifugation, and the mixture was further placed on ice by adding 0.1M CaCl ₂ , 0.05M MgCl ₂ solution and the expression vector pHC033 prepared in Example 4. These cells were centrifuged again and uniformly dispersed in the same solution (DNA Cloning Vol. I. Practical Approach, IRL press, 1985). All solutions and containers used above were used after cooling at 4 ° C.

Subsequently, 0.2 ml of the cell suspension obtained above was added to a petri dish coated with liquid LB medium containing 12.5 μg / ml of tetracycline and incubated at 37 ° C. overnight to obtain an E. coli JM101 transformant containing pHC033. The transformant was named Escherichia coli DW / HC-033, and on March 7, 1997, it was deposited with the Korea Microbiological Conservation Center (KCCM), an affiliated company of the Korean spawn corporation, an international depository. Deposited as -10956;

Example 6: Expression of Chimeric Proteins

The transformant Escherichia coli DW / HC-033 was inoculated in about 3 ml of the expression medium of Table 1 below, incubated overnight at 37 ° C. at 250 rpm, and 0.5 ml of this culture was inoculated again in about 50 ml of the expression medium of Table 1 below. Shaking was incubated at 37 ° C. at 250 rpm until the absorbance at 650 nm reached 1.8 to 2.0. Subsequently, 0.25 ml of 0.1M IPTG (isopropyl-β-D-thiogalactoside) was added thereto, and cultured at 250 ° C. for 24 hours at 37 ° C. to express recombinant protein, followed by centrifugation to obtain cells, and a buffer solution (0.1% Triton). (Triton) suspended in 10 mM Tris-HCl, pH 8.0 containing X-100, 2 mM EDTA and 1 mM PMSF, cells were disrupted, and the lysate was 12% SDS-PAGE (see Figure 4).

Table 1: Composition of Expression Badges

In FIG. 4, lane M is a molecular weight size marker, lane 1 is the result of electrophoresis of cell lysate immediately before IPTG induction, and lane 2 is the electrophoresis of lysate of cells cultured for 24 hours after IPTG induction. Each of the results is shown; The up arrow indicates the position of the chimeric protein fused to the Helicobacter pylori CagA and the Vibrio Correra A2 subunit, and the down arrow indicates the position of the Vibrio Correra B subunit protein. As shown in Figure 4, it can be seen that the chimeric protein is expressed in the transformant E. coli DW / HC-033.

As described and demonstrated in detail above, the fusion recombinant DNA of the CagA gene of Helicobacter pylori and the A2 / B subunit gene of the Vibrio correrai toxin gene was artificially designed to facilitate its expression and genetic manipulation, and also Helicobacter Industrial utility is due to the combination of genes that show excellent immunogenicity against pylori, which is stable to the environment within the stomach and provides proteins that can easily penetrate the gastric mucosa and promote the production of secretory immunoglobulins (sIgA). It can be maximized. Therefore, the chimeric protein expressed from such recombinant DNA can be used as an active ingredient in the production of a diagnostic reagent for Helicobacter pylori or a vaccine for the prevention or treatment of Helicobacter pylori related diseases, It can also be used to generate antibodies against.

Claims (8)

The antigenic protein CagA (CagA) gene of Helicobacter pylori and the A2 / B subunit gene of the Vibrio cholerae toxin gene are linked as follows. Recombinant DNA with Sequence or Functional Equivalent: A chimeric protein in which the CagA of Helicobacter pylori and the A2 / B subunit of Vibrio correia toxin are fused, having the following amino acid sequence translated from the recombinant DNA of claim 1 or a functional equivalent thereof: A recombinant expression vector comprising the recombinant DNA according to claim 1, wherein the CagA of Helicobacter pylori and the A2 / B subunit of Vibrio correrai toxin are fused to express a chimeric protein. Recombinant expression vector pHC033 comprising the recombinant DNA of claim 1 and having the following genetic map capable of expressing a chimeric protein fused with CagA of Helicobacter pylori and A2 / B subunit of Vibrio correrai toxin. Escherichia coli DW / HC-033 (KFCC-10956) transformed with the expression vector pHC033 of claim 4. A method for producing an electric chimeric protein comprising culturing the host microorganism transformed with the recombinant DNA of claim 1 and obtaining a chimeric protein in which the CagA of Helicobacter pylori and the A2 / B subunit of Vibrio correrai toxin are fused. . The method of claim 6, The transformed host microorganism is E. coli DW / HC-033 (Escherichia coli DW / HC-033) (KFCC-10956) characterized in that Method for producing chimeric protein. A chimera prepared by the method comprising culturing the host microorganism transformed with the recombinant DNA of claim 1 and obtaining a chimeric protein in which the CagA of Helicobacter pylori and the A2 / B subunit of Vibrio correrai toxin are fused. protein.