KR0154498B1 - Process for preparing hepatitis-b virus surface antigen pre-s1 - Google Patents

Abstract

A fusion protein of pre-S1 protein, the surface antigen of hepatitis B virus (HBV) of the present invention, and glutathione-S-transferse (GST) of Escherichia coli, encoding the fusion protein An expression vector comprising a gene, E. coli transformed with the expression vector, and a method for producing the HBV pre-S1 protein comprising culturing the same, the pre-S1 proteins of the present invention are produced in E. coli in water-soluble form and antigen Sex and immunogenic activity.

Description

Method for preparing non- (B) hepatitis virus surface antigen pre-S1

1 shows amino acid sustained salts of preS-119 and preS1-56 proteins and base salts of genes encoding them,

Figure 2a shows the structure of the expression vector for preparing a fusion protein of the preS1-119 protein and glutathione-S-transferase (GST),

Figure 2b shows the structure of the expression vector for preparing a fusion protein of preS1-56 protein and GST,

Figure 3a shows the supernatant and precipitate obtained by centrifuging the cell extracts with and without the induction of protein expression in 1PTG and E. coli transformed with the expression vectors of the present invention, respectively. Is the result of electrophoresis (SDS-PAGE) on a 12.5% SDS-polyacrylamide gel,

Figure 3b is the result of Western blotting the gel of Figure 3a,

Figure 4a is the result of electrophoresis of the fusion protein of preS1-119 and GST, the sample treated with the thrombin and purified preS1-119,

4b is a result of electrophoresis of fusion proteins of preS1-56 and GST, a sample treated with thrombin and purified preS1-56,

Figure 5a shows the antigenicity of the purified preS1-119 protein,

5b shows the antigenicity of the purified preS1-56 protein,

Figure 6a shows the immunogenic ability of purified GST-preS1-119 fusion protein and preS1-119 protein,

Figure 6b shows the immunogenic ability of purified GST-preS1-56 fusion protein and preS1-56 protein.

The present invention relates to a method for preparing pre-S1 (preS1) protein, which is the surface antigen of hepatitis B virus (HBV) of the present invention, and more particularly, to the pre-S1 protein and glutathione-S-transferase of HBV. (Glutione-S-transferase, GST) fusion protein, an expression vector containing the gene encoding the fusion protein, a method for producing HBV pre-S1 protein comprising culturing E. coli and E. coli transformed with the expression vector It is about.

HBV is a pathogen that invades humans and causes chronic and acute hepatitis and, if worse, causes cirrhosis and liver cancer. Vaccination against surface antigens is a common way to prevent HBV infection, and more effective hepatitis vaccines need to be developed for the treatment of newborn or fetuses born from increasing HBV immunity and HBV-positive mothers. Do.

Exposed to the surface of HBV of the pre-S1 protein (Heernamm, et al., J. Virol., 52, 39 (1984); Persing, et al., Science, 234,1388 (1986)), pre-S1 The 21st to 47th amino acid sites of the protein bind to HBV receptors in human hepatocytes (Neurath, et al., Cell, 46, 429 (1986); Pontisso, et al., 173, 552 (1989)). It has a neutralizing effect on HBV infection (Neurath, et al., Vaccine, 7,234 (1989)).

Therefore, the pre-S1 protein may be added to a vaccine against an existing S antigen (Neurath, et al., Vaccine, 7,234 (1989)) or used as a reagent for diagnosing the presence of infectious HBV from serum of chronic hepatitis patients. (Petit, et al., Hepatol., 11, 809 (1990)).

Since the pre-S1 protein is present in less than 10% of the HBV envelope protein, it is difficult to separate it from the serum of hepatitis carriers and the protein is too large for direct synthesis. Attempts were made to produce pre-S1 proteins by genetic recombination techniques, but proteins containing pre-S1 expressed in eukaryotic cells were not secreted (Persing, et al., Science, 234,1388 (1986); Kuroki, et. al., Mol. Cell Biol., 9, 4459 (1989); Biemans, et al., DNA Cell Biol., 10, 191 (1991). In addition, the pre-S1 protein is difficult to express directly in E. coli, and the fusion protein expressed by fusing the β-galactosidase gene with the pre-S2 antigen by using the lac promoter in E. coli is recovered in the state of inclusion body inclusion. Recovery was low (Lin, et al., J. Mek. Virol., 33, 181 (1991)).

Therefore, the present inventors have continued to develop a method for producing pre-S1 protein with high yield from E. coli by genetic recombination technology. As a result, the present inventors have developed an expression vector containing a fusion gene of GST gene and pre-S1 protein. The pre-S1 protein was recovered from the water-soluble fusion protein expressed in E. coli transformed with the expression vector in high yield to complete the present invention.

An object of the present invention is to provide an expression vector comprising a water-soluble fusion protein comprising a pre-S1 protein, which is an antigen of HBV, and a gene encoding the fusion protein.

Another object of the present invention comprises the production of E. coli transformed with the expression vector and the water-soluble fusion protein containing pre-S1 protein, which is easy to purify, and recovering the same. It is to provide a manufacturing method.

In accordance with the above object, the present invention provides an expression vector capable of expressing the fusion protein in E. coli in a water-soluble form, comprising a fusion protein of E. coli GST and HBV free-S1 protein, and a gene encoding the fusion protein. do.

According to another object of the present invention, in the present invention, the transformed Escherichia coli is cultured under conditions enabling expression of E. coli transformed with the expression vector and the gene encoding the fusion protein, followed by GST and HBV from cell lysate. A method of preparing a fusion protein of GST and HBV pre-S1 protein, comprising isolating a fusion protein of pre-S1 protein, is provided. In addition, the present invention provides a method for recovering only the HBV pre-S1 protein after cleaving the fusion protein prepared above with a protease to remove GST.

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

The expression vector of the present invention comprises a gene encoding the pre-S1 peptide including a binding site for H cell and T cell epitopes of HBV adr subtype pre-S1 antigen and HBV receptor, at the 3'-terminus of the GST gene of E. coli. Bound fusion genes. The pre-S1 peptide may be of any length as long as it contains a desired site, but should be one that can be stably expressed in E. coli as a fusion protein with GST. Pre-S1 peptide consisting of about 56 amino acids having high expression rate without containing an attack site or pre-S1 peptide consisting of about 119 amino acids which are less subjected to protease attack, and most preferably PreS1-56 or preS1-119 peptide shown in FIG. In the case of the pre-S1 protein consisting of about 90 amino acids, the cleavage rate by the protease is high and the size of the cleaved protein fragments is similar, which makes it difficult to separate.

The fusion gene encoding the fusion protein of the pre-S1 peptide and GST is a DNA nucleotide sequence encoding the pre-S1 peptide at the 3'-end of the GST gene and known DNA manipulation methods, for example, restriction enzyme cleavage, DNA It can be manufactured by ligation using a ligase, a PCR method, or the like, and can also synthesize whole DNA by chemical DNA synthesis. The fusion gene, when expressed, includes a base sequence that has an appropriate proteolytic site of attack between the GST and the pre-S1 peptide to facilitate separation of only the pre-S1 peptide after production of the fusion protein.

The expression vector including the fusion gene may be prepared by inserting the whole fusion gene into an appropriate expression vector or by first preparing a nucleotide sequence encoding the pre-S1 peptide, and then using the expression vector of the existing expression vector including the GST gene. It can be prepared by inserting at the 3'-end of the GST. For example, a well-known vector pHBV-315 (Kim Kang, Kor. Biochem. J., 17,70 (1984)) containing a surface antigen gene of HBV adr subtype was used as a template, and PCR was performed using a synthetic primer. By synthesizing the gene of pre-S1 protein and linking it to the 3'-terminus of the GST gene contained in a commercialized expression vector, eg, pGEX-2T (available from Pharmacia). An expression vector capable of expressing the fusion protein of E. coli in a dissolved state is prepared.

The E. coli strains, for example, E. coli DH5ⓐ, BL21 (DE3), XL1-Blue or JM109 are transformed with the expression vector, and then the transformants are transformed under conditions suitable for the growth of E. coli while maximizing expression of the fusion gene. By culturing, the GST fusion protein of the pre-S1 peptide is expressed. The expressed fusion protein was recovered by centrifugation from the cell extract obtained by crushing the cells, and then separated by glutathione affinity chromatography using glutathione agarose (Smith Johnson, Gene, 67, 31-40 (1988)). It can be easily recovered by purification. The fusion protein may be cleaved with an appropriate protease, for example thrombin, and then GST may be removed by the same method as above, and only the pre-S1 peptide may be isolated and purified.

In the present invention, the free-S1 fusion protein is produced in soluble form in Escherichia coli, thereby facilitating purification, thereby obtaining a pre-S1 peptide in high yield. The inventors have produced a fusion protein fused protein of maltose binding protein (MBP) and pre-S1 peptide in E. coli in a soluble form (see Korean Patent Application No. 94-6459), but the production of GST fusion protein is MBP fusion. Compared to the production of protein, there is an advantage that the purification is much easier.

The pre-S1 proteins produced by the present invention and fusion proteins comprising them can be used as antigens for the production of antibodies specific for HBV pre-S1, and can also be used for the production of vaccines that are more effective than conventional vaccines. . It will also be useful for detecting pre-S1 antibodies for diagnosing HBV infection.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited by the following examples.

Example 1

Primer 1 (5'-CGAGG ATCCA TGGGA GGTTG GTCTT CC-3 ') and Primer 2 (5'-GCTGG ATCCG GCCTG AGGAT GACTG TC-3') were synthesized to have a site cleaved by thrombin fused with GST, Using the known vector pHBV-315 (Kim Kang, Kor. Biochem. J., 17, 70 (1984)) containing the gene for the surface antigen of the HBV adr subtype as a template, the N- of the pre-S1 protein The DNA sequence encoding the terminal 119 amino acids was synthesized by the PCR method (Figure 1).

The PCR product was digested with restriction enzyme BamHI to obtain a 357 bp DNA fragment, which was linked to the 3′-terminus of the GST gene of the expression vector pGEX-2T (Pharmacia, USA) digested with BamHI to GST gene and HBV preS1. A plasmid pGSTpreS1-119 was constructed containing the fusion gene of the -119 gene (Figure 2).

E. coli DH5ⓐ transformed with the plasmid pGSTpreS1-119 was deposited on February 21, 1995, with the accession number KCTC 8646P to the Genetic Bank of Korea (KCTC).

Example 2

Using primers 1 (5'-CGAGG ATCCA TGGGA GGTTG GTCTT CC-3 ') and primer 3 (5'-GCTGA ATTCG TAGTA ATTTG CCTCT GGCCA ACG-3') to have a site fused with GST and cleaved by thrombin PCR was performed as in Example 1 except that the DNA sequence encoding 56 amino acids of the N-terminus of the pre-S1 protein was synthesized (FIG. 1).

The 173 bp DNA fragment obtained by cleaving the PCR product with restriction enzymes BamHI and EcoRI was linked to the 3'-terminus of the GST gene contained in the expression vector pGEX-2T cleaved with BamHI and EcoRI, and the GST gene and HBV preS1. The plasmid pGSTpreS1-56 containing the fusion gene of the -56 gene was constructed (Figure 2).

Escherichia coli BL21 (DE3) transformed with plasmid pGSTpreS1-56 was deposited on February 21, 1995 under the accession number KCTC 8645P to the Genetic Bank of Korea (KCTC).

Example 3

Using primer 1 (5'-CGAGG ATCCA TGGGA GGTTG GTCTT CC-3 ') and Primer 4 (5'-GAGGA ATTCC TGGCA CGGTT GTCAA TATGC C-3') to have a site that is fused with GST and cleaved by thrombin PCR was performed as in Example 1 except that the DNA sequence (HBV preS1-90 gene) encoding 90 amino acids at the N-terminus of the pre-S1 protein was synthesized.

The 270 bp DNA fragment obtained by cleaving the PCR product with restriction enzymes BamHI and EcoRI was linked to the 3'-terminus of the GST gene contained in the expression vector pGEX-2T digested with BamHI and EcoRI, and the GST gene and HBV preS1. A plasmid pGSTpreS1-90 containing a fusion gene of -90 gene was constructed.

Example 4

Using the plasmid pGSTpreS1-119 prepared in Example 1, according to the calcium chloride (CaCl ₂ ) method (Mamiatis et al., Molecular Cloning, A Laboratory Manual, pl82 (1991)), E. coli DHⓐ (available from Pharmacia, USA).

10 ml of overnight culture of the transformed strain was inoculated into 1 liter of nutrient liquid medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) containing 100 µg / ml ampicillin and incubated at 37 ° C for 2 hours. Then, IPTG (isopropy1-β-D-thiogalactoside) was added to a final concentration of 0.1 mM and incubated for 4 hours to induce the production of the fusion protein.

The cells were harvested by centrifugation of the culture medium before induction and 50 µl each after induction at 4,000xg for 10 minutes, followed by 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) method. The production of the fusion protein was confirmed by Western blotting using mouse monoclonal antibody F35.25 (manufactured by Immunotech) that specifically binds to the pre-S1 antigen (Fig. 3, column 3, 4). As a result, the fusion protein of GST and preS1-119 (hereinafter referred to as 'GSTpreS1-119') was expressed, but it was confirmed that some of them were cleaved in the cell near the 56th amino acid from the N-terminus of preS1.

Figure 3a is the result of 12.5% SDS-PAGE of each sample, Figure 3b is the result of Western blotting the protein sample of Figure 3a. In Figures 3a and 3b, column M is the standard protein molecular weight label, columns 1 and 2 represent samples before and after induction of E. coli containing the GST protein gene, respectively, and columns 3 and 4 are pGSTpreS1- respectively. Samples before and after induction of 119 are shown, and arrows 1 and 3 indicate the sizes of GSTpreS1-119 protein and GSTpreS1-56 protein, respectively.

To isolate the fusion protein resulting from the expression of pGSTpreS1-119, E. coli precipitate obtained by centrifuging 1 liter of the culture solution at 4,000 × g for 10 minutes was treated with Tris buffer 1 (50 mM tris-hydrochloric acid, 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid (EDTA), It was suspended in 0.1 ml of phenylmethylsulfonyl fluoride (PMSF), 0.02% NaN3, pH 7.5) 20, and then crushed by ultrasonic treatment twice for 5 minutes. The cell extract was centrifuged at 12,000 rpm (using Beckman JA20 rotor) for 10 minutes to obtain a supernatant.

The supernatant was loaded on a glutathione-agarose column (Sigma, USA) equilibrated with Tris buffer 1 and eluted with reduced glutathione 5 mM to obtain about 15 mg of fusion protein (Figure 4a, column 1). .

50 mg of the fusion protein was dissolved in 50 µl of digestion buffer (Tris-hydrochloric acid 50 mM, NaCl 150 mM, calcium chloride 2.5 mM, pH 8.0), 0.2 µl of thrombin was added and reacted at room temperature for 2 hours, and then the glutathione agarose column The protein solution, which was loaded and not bound to the column, was recovered to obtain 10 µl of 119 amino acid pre-S1 antigen (hereinafter referred to as 'preS1-119') (Fig. 4a, row 3). That is, 2 mg of preS1-119 was obtained from 1 liter of culture.

Example 5

As a result of repeating the same procedure as in Example 4 using E. coli BL21 (DH3) (Novagen) transformed with the plasmid pGSTpreS1-56 prepared in Example 2, the GSTpreS1-56 fusion protein was higher than the GSTpreS1-119 fusion protein. It was confirmed that it was expressed with high efficiency (Fig. 3, column 8), and confirmed that this fusion protein is present only in the soluble fraction of the cell extract (Fig. 3, column 9). From the cell extract, fusion protein was isolated and purified by glutathione agarose column chromatography. As a result, about 35 mg of fusion protein was obtained from 1 liter of culture medium (FIG. 4b, column 2) and passed through the glutathione agarose column. 5 mg of pre-S1 antigen (hereinafter referred to as 'preS1-56') of 56 amino acids was obtained (FIG. 4B and 3rd column).

Example 6

When E. coli DH5ⓐ was transformed with the plasmid pGSTpreS1-56 prepared in Example 2 and the same procedure as in Example 4 was repeated, the expression level of the antigen was 5 mg, which was not different from that of Example 5.

Example 7

As shown in Example 4 using the E. coli BL21 (DE3) (Novagen) transformed with the plasmid pGSTpreS1-90 prepared in Example 3, the fusion protein is produced as efficiently as the expression efficiency of pGSTpreS1-56 Confirmed. However, as in the expression of pGSTpreS1-119, the GSTpreS1-90 fusion protein was found to be partly cleaved by proteolytic enzymes in the cell and converted to GSTpreS1-56 size protein (Fig. 3, column 6). . In addition, the efficiency of degradation by proteolytic enzymes was higher than that of pGSTpreS1-119 (Fig. 3b, row 4 and row 6), so no further experiments were made for the preparation of preS1 antigen.

In FIG. 3, column 5 is a sample before inducing expression of pGSTpreS1-90, and column 6 is a sample after induction. Arrow 2 shows the size of the GSTpreS1-90 fusion protein. Isolation and purification of this fusion protein can be carried out as in Example 4.

Example 8

The antigenicity of the pre-S1 protein obtained above was confirmed by indirect ELISA reaction (Engvall Perlman, J. Immunol., 109, 129 (1972)). That is, the preS1-119 protein obtained in Example 4 and the preS1-56 protein obtained in Example 5 were coated on each well of the microplate by 0, 12.5, 25, 50, 75, 100, 150, and 200 ng, respectively. Mouse monoclonal antibody F35.25 (manufactured by Immunotech, France) (quantity: 10 ng or 20 ng) that specifically binds to the S1 protein was added, and then reacted at room temperature for 2 hours. The plates were washed with TBST solution (10 mM Tris, pH 7.5 150 mM NaCl, 0.05% Tween-20), and the amount of peroxidase-bound anti-mouse IgG antibody (peroxidase conjugated goat anti-mouse IgG, manufactured by Sigma) was 1 Dilute to 1000 and add and react at 37 ° C for 1 hour. After 100 μl of substrate solution (0.04% of o-phenylene diamine, 0.03% of hydrogen peroxide) was added, the reaction was stopped by adding 50 μl of 2.5M sulfuric acid. Absorbance was measured at 492 nm using an ELISA reader (Titertek Multiskan PLUS), and the results are shown in FIG. 5a and 5b show the antigenicity of preS1-119 and preS1-56, respectively.

As can be seen in Figure 5, as the amount of antigen increased as the antigen-antibody binding amount, it can be seen that both the 56 amino acid pre-S1 protein and 119 amino acid pre-S1 protein acted as antigen have.

Example 9

10 μl of preS1-119 and GSTpreS1-119 obtained in Example 4, and preS1-56 and GSTpreS1-56 obtained in Example 5, respectively, were injected into the 10 week old female mice (Balb / C) for 4 weeks at weekly intervals. 3 days after each injection, blood was collected from the tail. Procedure as in Example 8 using 200 ng of each protein as antigen and adding the blood diluted 1: 1000 instead of positive anti-mouse IgG antibody to confirm that antibody against preS1 was generated in mouse serum Was repeated to perform ELISA. The result is the same as in FIG. 6, and each antigen and the fusion protein both induced immunity.

Figure 6a is a result showing the immunogenic ability of preS1-119 and GSTpreS1-119 mice, Figure 6b is a result showing the immunogenic ability of preS1-56 and GSTpreS1-56. It is the result which showed the immunogenic ability of GSTpreS1-56 as a negative control group. Serum obtained by injecting mice with GST as a negative control was also shown by ELISA in the same manner.

As described above, the pre-S1 protein consisting of 56 amino acids or 119 amino acids purified from fusion proteins expressed in E. coli transformed with plasmids pGSTpreS1-119 and pGSTpreS1-56 showed antigenicity and immunogenic activity. .

Claims (12)

From the N-terminus of the pre-S1 protein of hepatitis B virus (HBV) to the 3'-terminus of the gene encoding the glutathione-S-transferase (GST) of Escherichia coli, from 50 to 58 amino acids E. coli transformed with an expression vector operable in E. coli, comprising a fusion gene prepared by concatenating a polynucleotide encoding a pre-S1 peptide, and then transformed E. coli under conditions that enable expression of the fusion gene. Method of producing a fusion protein of GST and HBV pre-S1 peptide, comprising culturing and then separating the fusion protein of the water-soluble GST and HBV pre-S1 peptide from the cell lysate. The method of claim 1, wherein the transformed E. coli is E. coli BL21 (DE3) / pGSTpreS1-56 (KCTC 8645P). The method of claim 1, further comprising the step of purifying the fusion protein by glutathione affinity chromatography of the supernatant obtained by crushing the cells after culturing the transformed Escherichia coli and then centrifuging. The fusion protein prepared by the method of any one of claims 1 to 3 is cleaved with thrombin, and glutathione affinity chromatography is used to remove glutathione-S-transferase and 50 from the N-terminus of pre-S1 protein. Hepatitis B virus pre-S1 peptide, characterized in that for separating only the pre-S1 peptide consisting of to 58 amino acids. A fusion protein in which a pre-S1 peptide consisting of 50 to 58 amino acids is fused from the N-terminus of glutathione-S-transferase hepatitis B virus pre-S1 protein. The fusion protein of claim 5, wherein the pre-S1 peptide has the following amino acid sequence: A fusion gene encoding a fusion protein of glutathione-S-transferase and a pre-S1 peptide consisting of 50 to 58 amino acids from the N-terminus of the pre-S1 protein of hepatitis B virus. The fusion gene according to claim 7, wherein the gene encoding the pre-S1 peptide of the hepatitis B virus has the following nucleotide sequence: An expression vector comprising the fusion gene of claim 7. The plasmid pGSTpreS1-56 of claim 9. E. coli transformed with the expression vector of claim 9. The method of claim 11, E. coli BL21 (DE3) / pGSTpreS1-56 (KCTC 8645P).