KR100210508B1 - Process for the preparation of vacuolating cytotoxin of helicobacter pyroli in e.coli - Google Patents

Abstract

The present invention relates to a method for preparing a vacuolating Cytotoxin (Vac), an antigenic determinant protein of Helicobacter pylori (H. pylori), in the form of a fusion protein using Escherichia coli, E. coli transformed with a Ub-V200 fusion protein expression vector comprising a fusion gene linked to a gene encoding a protein (V200) consisting of the 168th to 367th amino acids of Lori Vac, and suitable for expression of the fusion protein. Culturing the recombinant E. coli cells expressing the fusion protein, washing the insoluble precipitate with a buffer containing triton, dissolving with urea, gel filtration chromatography and heparin-sepharose chromatography. Diagnosis of H. pylori infection by the method of preparation of Ub-V200 fusion protein of the present invention The Ub-V200 fusion protein that can be useful for vaccine development can be mass-produced with high yield.

Description

Method for preparing vacuole cytotoxin protein of Helicobacter pylori from recombinant Escherichia coli

Figure 1 shows the procedure for cloning the fragment V200 of vacuole cytotoxin protein (Vac) of Helicobacter Philo into the E. coli expression vector pETUB-V200 of the present invention,

Figure 2 shows the results confirmed by the results of the modified polyacrylamide gel electrophoresis of the expression of the fusion protein ubiquitin-V200 of the present invention (a) and Western blotting using a ubiquitin single antibody (b),

Figure 3 shows the results of Western blotting using the modified polyacrylamide gel electrophoresis (a) and the serum of Helicobacter Philo infected after electrophoresis in each purification step of the fusion protein ubiquitin-V200 of the present invention (b) It is shown.

The present invention relates to a method for producing a vacuolating cytotoxin (Vac), which is an antigenic determinant protein of Helicobacter pylori (H. pylori) using Escherichia coli, Specifically, the fusion of 200 amino acids of the 168th to 367th amino acids of Vac, a specific antigen of Helicobacter pylori, reported as a causative agent of gastric and duodenal ulcers, fusion with Ubiquitin (Ub) It is expressed as protein (Ub-V200) to be useful for diagnosing H. pylori infection and developing vaccines.

H. pylori is an aerobic gram-negative spiral bacterium first isolated and cultured by Warren and Marshall in a biopsy of patients with duodenal ulcers (Warren and Marshall, Lancet, 1, 1273-1275 (1983)), originally Campylobacterfil Called Loridis (Campylobacter pyloridis), it was newly named Helicobacter pylori as it was identified as a new species by subsequent studies. H. pylori resides in the mucous layer that connects the gastric cavity of humans. Once incubated, H. pylori is parasitic for a lifetime and is found in high frequency in the gastric mucosa of patients with various gastrointestinal and duodenal diseases. It has been shown to be the underlying cause of duodenal ulcers (Alper J., et al., Science, 260, 159-160 (1993)), and has been reported to be a potential cause of stomach cancer (Parsonnet, et al., New Eng. J. Med., 325, 1127-1131 (1991); The Urogust Study Group, Lancet, 341, 1359-1362 (1993).

In 1994, a consensus conference hosted by the National Institute of Health (USA) defined gastric and duodenal ulcers as diseases caused by H. pylori, and treatment of eradication of H. pylori with antibiotics instead of conventional gastric acid regulator therapy. The recommendation for therapy was determined (NIH, NIH concensus development conference Helicobacter pylori in peptic ulcer disease (1994)). Indeed, various antibiotics currently being used have been shown to have an effect of over 90% on eradication of H. pylori and extremely low recurrence rate, which has been problematic in the treatment of gastric and duodenal ulcers.保 社, 1359, 22-23).

Accurate diagnosis is the basis for eradication of H. pylori. Among the many diagnostic methods that can diagnose infection, there are methods of penetrating and invading the body. Invasive methods of incorporating instruments into the body include biopsy of gastric tissue after endoscopy and histological identification of the presence of bacteria, and detection of urease enzyme activity in tissue after biopsy (Korean Patent Publication No. 94-6322 And culturing bacteria after biopsy. Noninvasive methods include indirectly measuring urease enzyme activity by taking urea labeled with ¹³ C or ¹⁴ C. However, these methods take a lot of time, it is difficult to carry out a large number of tests at the same time, there is a disadvantage that the repetitive test after treatment is unreasonable.

Because of the above problems, the method of diagnosing H. pylori infection using serum is preferred, which is due to the presence or absence of antibodies formed in the serum of H. pylori-infected humans using specific antigens of H. pylori. It is to judge whether or not. However, most of the antigens currently used are cell extracts of H. pylori or mixed proteins purified from them, which can be useful in developed countries with low infection rates. It is almost impossible to screen the infected person using a sample.

On the other hand, several types of H. pylori can be divided into 'type 1 H. pylori' that can express toxins and related proteins, and 'type 2 H. pylori' which does not express such proteins. In stomach and duodenal ulcer patients, only H. pylori type I is detected. Therefore, in countries where H. pylori infection is prevalent, such as Korea, it is more desirable to develop a diagnostic method that screens only those infected with H. pylori type I, who are more likely to develop stomach and duodenal ulcers. It is considered to be.

Therefore, while the present inventors are studying a method for diagnosing H. pylori infection of type 1, the present inventors have linked a part of the gene with the ubiquitin gene to use Vac as a specific antigen among proteins expressing only H. pylori type 1. By successfully mass-producing and purifying the fusion protein in Escherichia coli, the present invention has been completed.

An object of the present invention is a gene encoding a fusion protein of H. pylori vacuogenic cytotoxin protein (Vac), which can be used for diagnosis of infection and vaccine development of H. pylori infection, an expression vector comprising the same, and the expression vector It is to provide a transformant transformed into.

Another object of the present invention is to provide a method for preparing vacuole cytotoxin protein (Vac) of H. pylori in the form of a fusion protein.

In accordance with the above object, in the present invention, ubiquitin in the gene encoding the protein (V200) consisting of the 168th to 367th amino acid of Helicobacter pylori (H. pylori) vacuolating cytotoxin protein (Vac) Provided are a ubiquitin-vesicular cytotoxin protein (Ub-V200) fusion protein expression vector comprising a fusion gene linked to a gene, and E. coli transformed with the expression vector.

According to the above another object, in the present invention, E. coli transformed with an expression vector comprising the ubiquitin-vesicular cytotoxin protein (Ub-V200) fusion gene is cultured under conditions suitable for expression of the fusion protein, and the fusion protein is expressed. Preparing a Ub-V200 fusion protein comprising pulverizing the recombinant E. coli cells, washing the insoluble precipitate with triton-containing buffer, dissolving with urea, gel filtration chromatography and heparin-sepharose chromatography. Provide a method.

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

Ubiquitin used in the fusion protein of the present invention is a protein having a molecular weight of 8,500 Daltons consisting of 76 amino acids, present in all eukaryotic cells, but not in prokaryotic cells such as E. coli. When proteins are linked to ubiquitin and expressed in the form of fusion proteins, ubiquitin increases the expression rate of the linked protein or protects the protein from proteolytic enzymes (Sabin EA et al., Biotechnology, 7, 705-709 ( 1989). In addition, the expression of ubiquitin can be confirmed by using an anti-ubiquitin antibody, and can be usefully used for purification.

In order to prepare V200 as a fusion protein with ubiquitin according to the present invention, nucleic acid (DNA) is first extracted from H. pylori (ATCC 43504) and sequence information of Vac (Cover AL, et al., J, Bial. Chem , 269, 10566-10573 (1994); Telford, JL, et al., J, Exp. Med. 179, 1653-1658 (1994)) and the 5'-terminus of the V200 gene encoding the protein at the V200 site. A primer for polymerase chain reaction (PCR) corresponding to the 3'-end is designed and synthesized. These primers have a nucleotide sequence overlapping with the 3'-terminus of the ubiquitin gene synthesized in the present invention at the 5'-end, and the corresponding primer is inserted with an end codon and a restriction enzyme recognition site, thereby starting from the ubiquitin start codon. The protein is synthesized so that protein synthesis is completed at the artificially inserted end codon, and at the same time, it is easy to clone into an E. coli expression vector.

Using the primers, DNA of H. pylori is used as a template to amplify a gene of a desired region by a PCR reaction, and the amplified gene is combined with an artificially synthesized ubiquitin gene (Ub-V200). Subsequently, the Ub-V200 gene is amplified in a large amount by a PCR reaction, and the fragment obtained by cutting the amplified gene with a restriction enzyme is inserted into an appropriate E. coli expression vector to prepare a Ub-V200 fusion protein expression vector. E. coli is transformed with the recombinant vector, and then cultured under conditions suitable for expression of the fusion protein. Whether the protein is produced can be confirmed by the expression of Ub-V200 by Western blotting using a polyacrylamide gel electrophoresis, and then using a ubiquitin single antibody.

Escherichia coli transformed with an expression vector containing the recombinant Ub-V200 gene was shaken in Luria medium containing ampicillin and chloramphenicol and then transferred to the same culture, followed by shaking culture. The bacterial growth rate was 650. Absorbance at 0.3 is about IPTG (Isopropyl- -D-thiogalactopyranoside) is added to induce the expression of Ub-V200. The E. coli cells were precipitated by centrifugation of the culture medium, and then suspended in Buffer A (50 mM Tris, pH 8.0, 0.1 M NaCl, 1 mM DTT, 1 mM EDTA, 1 mM PMSF), and the cells were crushed under pressure using a French press. Centrifuge. Remove the soluble protein and suspend the cell precipitate in Tris buffer containing Triton X-100, then stir and centrifuge to collect only the cell precipitate. The insoluble precipitate containing Ub-V200 is suspended with a buffer solution containing urea, stirred at room temperature and then centrifuged. Take the homolog and dilute with buffer containing 4M urea and flow it into Sephacryl S-200 gel filtration column previously equilibrated with the same buffer, then elute with the same buffer and transfer SDS-polyacrylamide gel to transfer Ub Collect only fractions that contain -V200. The collected solution was passed again through the previously equilibrated heparin-sepharose with the same buffer solution, and a protein fraction was collected by adding a buffer of 0M to 0.5M sodium chloride concentration gradient, and the portion was subjected to SDS-polyacrylamide gel transfer and subjected to Ub. By collecting only the part of the -V200 fusion protein, a high purity Ub-V200 fusion protein can be obtained.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following Examples are only for illustrating the present invention, but the scope of the present invention is not limited to these.

Example 1

Amplification of the V200 gene, part of the Vac gene, from H. pylori DNA

[Step 1]

DNA was extracted from H. pylori (ATCC 43504) according to Schleiff's method (Scglief RF, et al., Practical Methods in Molecular Biology, p98 Springer, New York (1981)). That is, inoculated in Brucella Broth, Difco Cat No. 0495-01-1, to which 5% fetal bovine serum of H. pylori strain was added, and then anaerobic jar (Difco Cat No. 1951-30). -1) was incubated for 4 to 5 days at 37 ℃ in the presence of 5% O ₂ and 10% CO ₂ . The cell precipitate was dissolved in GES solution (60% guanidium thiocyanate, 0.1M EDTA, 0.5% sarcosyl), extracted with the same volume of phenol / chloroform solution, and the whole DNA was precipitated with isopropanol. This precipitate was dissolved in 100 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).

[Step 2]

The primers PVACT2 and PVACBAML were synthesized from previously known sequencing information of the V200 gene (Cover A. L., et al., J. Bial. Chem., 269, 10566-10573 (1994)):

or

Primers PVACT2: 5'-CTTGGTGTTGAGACTCCGCGGTGGTTTAGAAATCAATAACGTCG-3 ' Designed to have the same nucleotide sequence as 20 nucleic acids).

Primer PVACBAML: 5'-AAAAAGGATCCGTCGACTAATCGGCTTTAGTGTTGAT-3 '(having a termination codon to terminate detoxification after the 367th codon of Vac and at the same time have restriction sites SalI and BamHI recognition sites).

[Step 3]

The reaction tube was templated with 1 ng of H. pylori DNA obtained in step 1, and the primer PVACT2 2 was used. And primer PVACBAML 2 Insert 10 10-fold polymerization buffer (500 mM KCl, 100 mM Tris-HCl, pH 9.0, 1.0% Triton X-100), 10 μl of 2 mM dNTP (2 mM dGTP, 2 mM dCTP, 2 mM dATP and 2 mM dTTP), 2.5 monomer Taq polymerization Enzyme and distilled water were added so that the total volume was 100 μl. PCR was performed 40 times under the conditions of 1 minute (denatured step) at 95 ° C, 40 seconds (annealing step) at 55 ° C, and 2 minutes (synthetic step) at 72 ° C. Was carried out. The PCR product obtained above was separated from a 5% polyacrylamide gel to confirm that approximately 600 base pairs of DNA were long and separated and purified from agarose gel under the same conditions (hereinafter, this DNA fragment was referred to as 'fragment V200'). .

Example 2

Synthesis of the Ubiquitin Gene

[Step 1]

The primers UBI1, UBI2, and UBI3 were synthesized from known base sequence information of the following ubiquitin gene (Ozkaynak, et al., EMBO, J., 6, 1429-1439 (1987)):

or

[Step 2]

Three oligonucleotides synthesized in step 1, ie, primer UBI1 2, were added to the reaction tube. , Primer UBI2 2 And primers UBI3 2 10 μl of 10-fold polymerization buffer, 10 μl of 2 mM dNTP, 2.5 units of Taq polymerase and distilled water were added to make the total volume 100 μl, and the PCR reaction was carried out in the same manner as in Step 3 of Example 1. Was carried out. The PCR product thus obtained was separated on a 5% polyacrylamide gel to extract and purify about 270 base pairs of DNA (hereinafter, this DNA fragment is referred to as 'fragment Ub'), and then dissolved in 20 µl of TE solution.

Example 3

Preparation of V200 Gene Expression Vector

Plasmid pET11C purchased from Novagen (Novagen Inc., Madison WI 53711, USA) 2 Was digested under the conditions of NEB buffer 3 (10 mM Tris-HCl, 10 mM MgCl ₂ , 100 mM NaCl, 1 mM dTT, pH 7.0) with restriction enzymes Ndel and BamHI, and then separated by 0.7% agarose gel to obtain a 5.8 kb fragment. Separation and purification (hereinafter, this fragment is referred to as 'fragment PNB').

On the other hand, since the 5'-end of the fragment V200 gene obtained in Example 1 and the 3'-end of the fragment Ub obtained in Example 2 overlapped with 25 bases, they were combined by PCR as follows. Fragment V200 10ng, Fragment Ub 10ng, primer UBI1 2 in reaction tube And primer PVACBAML 2 10 μl of 10-fold polymerization buffer, 10 μl of 2mM dNTP, 2.5 unit Taq polymerase and distilled water were added to make the total volume 100 μl, and the PCR reaction was carried out in the same manner as in Step 3 of Example 1. . The PCR products obtained above were completely cleaved with the restriction enzymes Ndel and BamHI under the conditions of NEB buffer 3, and then extracted with phenol / chloroform and dissolved in 20 µl of TE solution. This is referred to as 'Short UB-V200-N / B'. In the condensation reaction tube, add 100 ng fragment UB-V200-N / B and 100 ng fragment PNB, 2 μl of 10-fold conjugation reaction solution, 10 units of T4 ligase and distilled water to give a total volume of 20 μl. The reaction was carried out at 16 ° C. for 12 hours. After the reaction, the E. coli HB101 (ATCC 33694) competent cells were transformed to obtain an expression vector pETUB-V200 containing fragment UB-V200, which was then expressed again in E. coli BL21 / (DE3) / pLys, S (Novagen). Inc., Madison WI53711, USA).

1 shows the cloning of fragment V200 of H. pylori vacuole cytotoxin protein (Vac) into E. coli expression vector pETUB-V200.

The transformed Escherichia coli BL21 / (DE3) / pLys, S / pETUB-V200 was deposited on January 9, 1996 to the gene bank attached to the Biotechnology Research Institute of Korea Institute of Science and Technology as Accession No. KCTC 0227BP.

Example 4

Induction of Expression of the V200 Gene from Escherichia Coli

E. coli strain transformed in Example 3 50 Of Ampicillin and 34 Of Shaken for 12 hours in Luria medium (6% bactotriptone, 0.5% yeast extract, 1% sodium chloride) containing chloramphenicol, followed by 1 100 Was transferred to Luria medium containing ampicillin and chloramphenicol, and shaken and cultured at 37 ° C. for about 4 hours to obtain IPTG (Isopropyl-) at a bacterial absorbance of about 0.3 at 650 nm. -D-thiogalactopyranoside) was added to a final concentration of 0.5 mM. Immediately before addition of IPTG and after 1, 3, 5 and 7 hours of addition respectively 10 Cell precipitates were collected by centrifugation at 10,000 x G for 15 minutes each. The collected cell precipitate was subjected to 10% SDS-polyacrylamide gel electrophoresis according to the method of Laemmi et al., Nature 227, 680 (1970), and then the protein was purified by Coomassie Brilliant blue R250. Stained.

Subsequently, western blotting is performed using the ubiquitin monoantibody with the protein isolated on the gel. First, the protein isolated on the gel was subjected to nitrocellulose membrane (Bio-Rad Lab., Pore size 0.22) according to Tobin's method (Towbin, et sl., Proc. Natl. Acad. Sci. USA 76, 4750 (1979)). Μl, CA, USA) and the membranes were placed in PBS (10 mM phosphoric acid, pH 7.0, 0.15 M sodium chloride) containing 0.5% Tween 20 and crushed with gentle shaking for 2 hours at room temperature. Subsequently, 1000-fold dilution of ubiquitin monoantibody was added to PBS containing 0.5% gelatin and 0.05% Tween 20, and reacted with gentle shaking for 1 hour at room temperature, followed by washing several times with PBS containing 0.2% Tween 20 for 5 minutes. . Subsequently, anti-human immunoglobulin G (Bio-Rad Lab, anti-human IgG-HRP, 0.01 mg, CA USA) labeled with horseradish peroxidse contains 0.5% gelatin and 0.05% Tween 20 Diluted 1: 500 times with diluted PBS, then shaken for 1 hour at room temperature, washed 4 times with PBS containing 0.2% Tween 20 for 5 minutes, and then washed twice with 50 mM Tris buffer (pH 7.0). It was. 400 stop this Of Was developed by adding 50 mM Tris buffer solution containing 4-chloro-1-naphthol and 0.03% hydrogen peroxide solution.

Figure 2 shows the results of confirming the expression of the fusion protein ubiquitin-V200 by denatured polyacrylamide gel electrophoresis (A) and Western blotting (B) using ubiquitin single antibody.

In Figures 2 (a) and (b), column m represents the standard protein molecular weight, column 1 represents the cell extract immediately before application of IPTG, and columns 2, 3, 4 and 5 represent IPTG. Cell extracts obtained by incubating for 1, 3, 5 and 7 hours after addition are shown respectively.

Example 5

Tablets of Ub-V200

[Step 1]

Culture process of E. coli cells

Recombinant strain (BL21 / (DE3) / pLys, S / pETUB-V200) transformed with the expression vector pETUB-V200 containing the synthesized Ub-V200 fusion gene was prepared in Luria medium containing empicillin and chloramphenicol. After shaking for 12 hours, 5 Was transferred to Luria medium containing 1 L of ampicillin and chloramphenicol and shaken at 37 ° C. for about 3 to 4 hours. When bacteria grow at about 650 nm and absorbance is around 0.3, IPTG (Isopropyl-) -D-thiogalactopyranoside) was added to a final concentration of 0.5 mM to induce the expression of recombinant Ub-V200. About 3 hours after the addition of IPTG, the cell culture was finished and E. coli cell precipitates were collected by centrifugation at 5,000 × G for 30 minutes.

[Step 2]

Cell fusion and purification of Ub-V200 fusion protein

E. coli cells expressing Ub-V200 fusion protein 2.5g 30 Suspended in Buffer A (50 mM Tris, pH 8.0, 0.1 M NaCl, 1 mM DTT, 1 mM EDTA, 1 mM PMSF), crushed cells under pressure using a French press, and centrifuged for 15 minutes at 10,000 x G. It was. Removing soluble protein and obtaining insoluble precipitate 30 After suspension in 50 mM Tris (pH 7.5) buffer containing 1% of Triton X-100, the precipitate was collected by stirring at room temperature for 15 minutes and then centrifuging at 10,000 × G for 30 minutes.

Insoluble precipitate containing Ub-V200 5 containing 8M urea Suspended with buffer solution B (20 mM sodium phosphate (pH 7.2), 1 mM EDTA, 1 mM DDT), stirred at room temperature for 1 hour, and then centrifuged at 10,000 x G for 30 minutes to remove insoluble precipitate and supernatant. Got it. The supernatant was diluted with buffer B so that the urea concentration was 4M, then injected into a Sephacryl S-200 gel filtration column previously equilibrated with buffer B containing 4M urea, and eluted with the same buffer. SDS) -polyacrylamide gel electrophoresis to collect only the Ub-V200 fusion protein portion. The collected fractions were again equilibrated with buffer B containing 4M urea (1.0 in heparin-sepharose) × 1.0 ) And 80 containing a concentration gradient of 0M to 0.5M sodium chloride Buffer B was added to the isolated protein fraction. Collected each. Collection SDS-polyacrylamide gel electrophoresis to collect only the portion containing the Ub-V200 fusion protein, Ub-V200 fusion protein of more than 95% high purity was obtained.

FIG. 3 shows the results of Western blot using (a) modified polyacrylamide gel electrophoresis in each purification step of the fusion protein ubiquitin-V200 and the blotting of Helicobacter pylori infected person after electrophoresis. .

In column (a) and (b) of FIG. 3, column m represents the standard protein molecular weight, column 1 represents total cell homogenate after cell disruption, and column 2 shows the protein dissolved in 8M urea. The third column shows the protein solution after Sephacryl S-200 gel filtration chromatography, and the fourth column shows the purified Ub-V200 protein after heparin-Sepharose chromatography.

Claims (6)

A fusion gene in which a ubiquitin gene is linked to a gene encoding a protein (V200) consisting of the 168th to 367th amino acids of Vacuolating Cytotoxin (Vac) of Helicobacter pylori (H. pylori). A ubiquitin-vesicular cytotoxin protein (Ub-V200) fusion protein expression vector comprising the fusion gene of claim 1. The expression vector pETUB-V200 according to claim 2. E. coli transformed with the expression vector of claim 2. E. coli BL21 / (DE3) / pLys, S / pETUB-V200. E. coli is cultured under conditions suitable for the expression of the fusion protein, recombinant E. coli cells expressing the fusion protein are pulverized, the insoluble veteran is washed with a buffer containing Triton, and then dissolved in urea and gel filtration. Chromatography and heparin-sepharose chromatography method for producing a Ub-V200 fusion protein comprising the step.