CN1144272A - Novel Hepatitis B Surface Antigen Containing Immunological Determinants of Presurface Antigen 1 and 2 - Google Patents

Abstract

The present invention relates to a new-type hepatitis B surface antigen which is structured by gene-engineering technology and at the same time contains front surface antigen S1 and S2 immune determinants and S antigen. This new-type gene structure can stably secrete and represent the fused particles having triple antigenicity of front S1, front S2 and S simultaneously in the recombined virus vaccinicum system. These fused particles can be used as new-type antigen for preventing and curing hepatitis B, and the coded new-type antigenic fused gene also can be directly used for nucleic acid immunotherapy of hepatitis B.

Description

Novel Hepatitis B Surface Antigen Containing Immunological Determinants of Presurface Antigen 1 and 2

Hepatitis B virus is the main infectious virus that causes liver disease in humans. Many hepatitis B patients recover after an acute stage, and many people turn to chronic and become chronic hepatitis. There are more than 100 million hepatitis B virus carriers in my country.

Universal vaccination against hepatitis B is an important measure to control infection. The blood-derived vaccine is made by separating the hepatitis B surface antigen particles in the patient's serum. Due to the difficult source, high cost and poor safety, it is no longer produced in developed countries. Using genetic engineering means to express hepatitis B surface S antigen in yeast, mammalian cells, recombinant vaccinia and other systems, and use it as a genetic engineering vaccine, which has the advantages of safer, economical and effective, but its immunogen is basically the same as that of blood-derived vaccines . There are still some deficiencies, such as: (1) About 10% of the population has no response or low response. (2) The required dosage is relatively large. (3) Unable to deal with immune escape variants. Therefore, foreign countries have been developing efficient and economical new hepatitis B vaccines.

HBsAg consists of three proteins, major protein (S), medium protein (M), and large protein (L), all three proteins are encoded by the DNA of the same reading frame in HBV, translated from different start codes ATG . The difference between M protein and S protein is that there are 55 more amino acids at the amino terminal of M protein, namely the pre-surface antigen 2 (preS2) region, while L protein has 108 or 119 more amino acids at the amino terminal of M protein, namely pre-surface antigen 1 (preS1) region. The M and L proteins are mainly present in the coat of the virus particle.

The PreS1 region contains a different antigenicity from S. There are immune determinants of T cells and B cells, and the peptides at the 21-47th amino terminal have binding sites for hepatocyte receptors (Neurath A.R et al., Cell 1986, 46, 429-436). It has the ability to induce the production of antibodies that neutralize the virus, thereby blocking the combination of the virus and liver cells to protect the body from infection. The L protein also activates preS1-specific T cells to overcome unresponsiveness to the S and M proteins. However, L protein is not easy to be secreted from cells, and it also inhibits the secretion of S protein. (Persing P.H. et al., Science 1986:234, 1388). In addition, since the L protein contains multiple sensitive sites for proteases and is easily degraded, it is difficult to obtain a large amount of L protein for use as a vaccine.

Studies with synthetic preS1 (21-47) polypeptides have confirmed that this region contains the site necessary for the binding of HBV to liver cells. Anti-preS1 monoclonal antibodies MA18/7 and F35.25 can effectively inhibit the binding of HBV to liver cells, with Ability to neutralize viruses (Petit M.A et al. Virology 1990: 180, 483-491). Immunization of orangutans can make it resistant to HBV challenge (NeurathA.R. et Vaccine 1989, 7, 234-236). However, if synthetic peptides are used as vaccines, there is still a long way to go before practical use due to low immunogenicity.

The preS2 region also contains different antigenicity from S, the preS2(120-132) region contains the recognition site of T cell immunity, and the preS2(133-143) contains the main recognition site of B cell immunity. Among them, the polypeptide at positions 136-147 contains the binding site of hepatocyte polyalbumin receptor (KroneB. et al. Hepatology 1990, 11, 1050-1056). HBV virus may cause infection of liver cells by combining with liver albumin. The antibody produced by immunizing animals with synthetic highly immunogenic polypeptide preS2 (120-145) also has the effect of neutralizing HBV virus (Neurath A.R. Vaccine 1986 4:35-37) (Milich.D.R.etal., J. EXP. Med, 1986, 16:532). Neurath et al. and Agata et al. reported that preS2 (120-145) is likely to be another recognition site of the hepatocyte receptor (NeurathA.R.et al., Cell, 1986, 46:429-436; Agata B.et al ., J. Virol., 1995, 69, 840-848). Using synthetic peptides as vaccines has poor immunogenicity and is far from practical application. However, the natural M protein is also extremely unstable, and it is difficult to obtain large quantities. Recent studies have shown that the preS2 region contains multiple sensitive sites to proteases, so that it is easily degraded by proteases during the preparation process. Studies have also shown that the two amino acid regions 1-13 and 20-46 in preS2 (namely, 120-132 and 139-165 calculated from the preS1 sequence) contain information that is conducive to the secretion of M protein from the cell to the cell. The transmembrane structure of (VonHeijne, Eur.J.Biochem 1988, 103, 431-438).

Utilize hepatitis B surface antigen to make carrier protein recently, successfully with exogenous gene fragment as, human herpes simplex virus type 1 HSV-1 (Valenzuela P. etc. Bio/Technology1985,3,323), human immunodeficiency virus HIV (Michel M.L. etc. Proc.Natl.Acad.Sci.U.S.A.1988 85,7957), poliovirus Polio (Delpeyroux F. et al. Science 1986,233,472) is inserted in the PreS2 region, respectively in yeast or mammalian cells to be expressed.

However, there has been no report of simultaneous fusion of different antigenic determinant genes at the 5' and 3' ends of the S gene.

The purpose of the present invention is to provide a hepatitis B recombinant antigen with a new structure that can block hepatitis B virus infection. The new antigen can be passed through a recombinant vaccinia virus (Vaccinia) containing the fusion gene of the hepatitis B surface antigen S gene, the preS1 epitope gene containing the hepatocyte receptor binding site, and the preS2 epitope gene containing both T cell and B cell epitopes. Virus), stably express and secrete fusion particles of hepatitis B S surface antigen and preS1, preS2. The particles have triple antigenicity of hepatitis B HBsAg, preS1 and preS2 and good immunogenicity predicted. The present invention can produce fusion particles of HBsAg, preS1 and preS2 through a large amount of recombinant vaccinia virus secretion, and after separation and purification, a safer and more efficient novel genetic engineering particle vaccine can be produced, and the recombinant vaccinia virus can also be used to prepare new hepatitis B live vaccines.

Using PCR technology, we artificially synthesized the nucleotide sequence corresponding to amino acid positions 21-47 in preS1, and removed 20 amino acids at the N-terminal of preS1 that may affect the secretion of S antigen. At the 3' end of the hepatitis B S surface antigen gene, it is fused with the synthetic preS1 nucleotide fragment at the corresponding amino acid 223 position.

In addition, the nucleotide sequence corresponding to amino acid 120-146 of the preS2 gene was artificially synthesized by PCR method, the protease sensitive site was removed as much as possible, and the S surface antigen containing both T cell and B cell epitopes was retained. order of secretion. At the 5' end of the hepatitis B S surface antigen gene, the first position corresponding to the amino acid sequence is fused with the synthetic preS2 nucleotide fragment. A fusion gene containing preS2 (120-146) fused at the 5' end of the HBsAg S gene and preS1 (21-47) at the 3' end was obtained. By constructing a recombinant vaccinia virus containing the fusion gene, triple antigenic particles carrying HBsAg and preS1 and preS2 were successfully expressed. Experiments have proved that this design scheme does not affect the formation of HBsAg particles and secretion from cells, nor does it affect the antigenicity of HBsAg. On the contrary, it is more conducive to the secretion from cells, and the formed particles also have the antigenicity of preS1 and preS2.

The fusion gene can also be expressed in eukaryotic systems such as mammalian cells (such as CHO) and yeast, and the basic properties of the expression product should be the same as those in recombinant vaccinia.

The invention provides HBsAg with a novel structure in which the amino terminal of HBsAg main protein is fused with the preS2 antigenic determinant, and the carboxy terminal is fused with the preS1 antigenic determinant. The novel antigen can be

preS2(120-146)；S

(1-223)。TK---thymidine kinase; P7.5---vaccinia promoter 7.5; MC---multiple cloning site; E---EcoRI; B---BamHI; H---HindIII; N--- NcoI; A---AccI; Lig---link; S1 preS1(21-47);S2

preS2(120-146);S

(1-223).

Figure 4: Southern blot hybridization identification of recombinant vaccinia virus vS2SS1 DNA, ECL image.

1. Tiantan strain; 2. pGJP/S2SS1; 3. vS2SS1; 4. Enter the HindIII enzyme cut molecular weight standard

Figure 5: Western blot hybridization identification of recombinant vaccinia virus vS2SS1 expression product, ECL image.

A. Hybridization with HBsAg monoclonal antibody; B. Hybridization with preS1 monoclonal antibody; C. Hybridization with preS2 monoclonal antibody

1. Tiantan strain; 2. vTH-2; 3. vS2SS1

preS1

preS2

Figure 6: Density ultracentrifugation of recombinant vaccinia virus vS2SS1 expressing triple antigen particles. HBsAg

preS1

preS2

Figure 7: Electron microscope observation photos of particles (scale length is 100nm).

Content of the present invention is specifically described in the following examples:

Materials and methods:

a. Restriction enzymes and ligases used in gene recombination are Boehringer products. Cell culture medium, DMEM/HG high glucose medium, fetal calf serum (FCS), etc. are all GIBCO products. 5-bromodeoxyuridine (BUdR) is a Fluka product. PCR kit and Taq DNA polymerase are Promega products.

b. Thymidine kinase (TK) gene-deficient cells used, human TK-143 cells (provided by American NIH Professor B.Moss), were cultured in DMEM medium containing 25 μg/ml BUdR and 5% fetal bovine serum . The vaccinia virus Tiantan strain (Vaccinia Virus, TianTan) and Vero cells were obtained from the Beijing Institute of Biological Products, Ministry of Health.

c. Cell transfection: Vero cells were transfected with calcium phosphate co-precipitated DNA (containing 10 μg plasmid and 10 μg protist DNA) after being infected with vaccinia virus Tiantan strain for 2 hours. Incubate at 37°C for 5 hours, suck off the transfection solution, add fresh culture solution, continue to culture for 48 hours, and harvest the cells. A recombinant vaccinia virus was constructed and obtained through stable secreted expression, that is, fusion particles of HBsAg main protein and preS1 and preS2 proteins. Specific methods include:

1. The preS2 antigenic determinant containing both T cell and B cell epitopes corresponding to the (120-146) nucleotide fragment and the PCR synthesis of the S gene and its fusion clone. (pWR/S2S)

2. PCR synthesis of the nucleotide fragment corresponding to the preS1 antigenic determinant (position 21-47) containing the hepatocyte receptor binding site and the clone fused with PWR/S2S. (pWR/S2SS1)

3. Construction of recombinant vaccinia expression plasmid containing triple fusion gene. (pGJP/S2SS1)

4. Construction and screening of recombinant vaccinia virus containing hepatitis B S gene and preS1 (21-47) and preS2 (120-146) genes. (vS2SS1)

5. Propagation of recombinant vaccinia virus, expression of HBsAg with preS1 and preS2 and identification of the product. (pS2SS1)

The schematic illustration of the present invention is as follows:

Figure 1: Schematic diagram of PCR synthesis of preS2 (120-146) epitope containing polyalbumin binding site and S gene and construction of its fusion clone pWR/S2S.

Explanation of various symbols:

5′端引物；3′ 3′端引物；引物2 preS2引物；引物B

HBsAg引物；S2 preS2(120-146)；S (1-226)；B---BamHI；H---HindIII；N---NcoI；A---AccI；Lig---连接。5'

5' end primer; 3' 3′ end primer; primer 2 preS2 primer; Primer B

HBsAg primer; S2 preS2(120-146);S (1-226); B---BamHI; H---HindIII; N---NcoI; A---AccI;

Figure 2: Schematic diagram of the PCR synthesis of the preS1(21-47) epitope gene containing the hepatocyte receptor binding site and the construction of the fusion clone PWR/S2SS1.

5′端引物；3′

3′端引物；S1 preS1(21-47)；S2

preS2(120-146)；S (1-223)； (1-226)；B---BamHI；H---HindIII；N---NcoI；A---AccI。5′

5' end primer; 3'

3′ end primer; S1 preS1(21-47);S2

preS2(120-146);S (1-223); (1-226); B---BamHI; H---HindIII; N---NcoI;

Figure 3: Schematic diagram of the construction of the recombinant vaccinia expression plasmid (pGJP/S2SS1) containing the S2SS1 triple fusion gene.

d. Preparation of plasmid DNA. Purified by Sepharose 2B column chromatography with alkali denaturation method.

e. Recombination of plasmids and bacterial transformation. Carry out with reference to the routine method on Sambrook J. et al.'s " molecular cloning " book, and the recipient bacterium used is TG-1.

f. Screening of recombinant vaccinia virus and extraction of viral DNA. For the preparation of vaccinia DNA, refer to the method in the literature (Esposto J. et al. J. Vivol. Meth: 1980, 2, 175). According to the method of Weir et al. (Weir J.P. et al., Proc. Natl. Acd. Sci. U.S.A. 1982, 79, 1210), the TK-recombinant virus selected plaques in the presence of BUdR, and analyzed and identified them.

g. Determination of HBsAg, preS1, preS2 antigens. For the determination of HBsAg, use the Auszyme kit (product of Abbott) or the detection kit of Shanghai Kehua Industry, and dilute the sample 1:10. For the determination of preS1 antigen, use the enzyme-linked detection kit of Alpha Company of our institute, use the double sandwich method, first coat the plate with anti-HBsAg polyclonal antibody, dilute the sample 1:10, and use the preS1(21-47) monoclonal antibody enzyme-linked conjugate to act . The principle of preS2 antigen detection is the same as preS1 double sandwich method, anti-preS2 monoclonal antibody Q19/10 is coated on the plate, and anti-HBsAg enzyme-linked conjugate is used for detection. The preS2 monoclonal antibody (Q19/10) used was provided by Dr. W.H. Gerlich, Germany. The antigen secreted into the culture medium can be measured directly with the culture medium. Determination of intracellular antigens: After the cells were suspended in PBS, they were frozen and thawed four times with liquid nitrogen, and the supernatant after centrifugation was a sample of the freeze-thawed cell lysate, which was sampled for determination.

h. Protein electrophoresis analysis of expression products. SDS polyacrylamide electrophoresis was carried out according to the Laemmli method (Laemmli U.K. et al., Nature, 1970, 223, 680).

i. ECL non-radioactive labeling detection is used for Southern blot and Western blot. Reagents and labeling procedures were performed using the procedures provided by Amersham.

The primary antibodies used in Western blot analysis were anti-HBsAg monoclonal antibody (H166, provided by Prof. Hofschneider, Max Planck Institute of Biochemistry, Germany) at 1:300 dilution. Anti-preS1 monoclonal antibody (125E11, provided by Professor Zhang Zuchuan, Shanghai Institute of Biochemistry), diluted 1:1000. Anti-preS2 monoclonal antibody (25-19, provided by Professor Hofschneider, Max Planck Institute of Biochemistry, Germany), diluted 1:6000.

The specific steps for implementation are described step by step below:

Example 1: PCR synthesis of preS2 (120-146) antigenic determinant gene and S gene containing both T cell and B cell epitopes and construction of its fusion clone.

The selected adr subtype hepatitis B virus contains the preS2 antigenic determinant of both T cell and B cell epitopes, the amino acid sequence number is 120-146, and the recombinant plasmid pADR-1 containing preS2 (Wu Xiangfu et al., Chinese Science, Series B, 1983 , 2:162-167) as a template, the preS2 DNA fragment containing the 120-146 position was amplified by PCR method. The 5' end primers used were:

5'GTCATCCTGGATCCATGCAGTG3'

BamHI

The total length is 22 nucleotides, and the underlined part is a BamHI restriction site designed and introduced at the 5' end of preS2. The 3' end primer is:

5′GTCCCGGCCATGGAGCCACC3′

NcoI

The total length is 20 nucleotides, and the underlined part is an NcoI restriction site designed and introduced at the 3' end of preS2. For the PCR and reaction system, see the methods provided by Promega. The product synthesized by PCR was double-digested with BamHI and NcoI to obtain a fragment containing preS2 (120-146) nucleotides for later use. In order to make the reading frame correct after the fusion of preS2 and the S gene, we also synthesized the nucleotide sequence of the S gene containing the adr subtype by PCR method, corresponding to amino acid sequence number 1-226. The 5' end primers used were:

5′GCACCGACCATGGAGAGCAC3′

NcoI

The total length is 20 nucleotides, and the underlined part is an NcoI restriction site designed and introduced at the 5' end of the S gene. The 3' end primers used were:

5′GGAGGTGTGGATCCGAGAGAG3′

A total of 21 nucleotides in length.

The PCR reaction system was the same as the synthesis of preS2 above. The PCR product was double-digested with NcoI and HindIII (the 3' end of the S gene contains a HindIII restriction site, see Figure 1) to obtain a nucleotide fragment containing S (1-226). In addition, the nucleotide fragment containing preS2 (120-146) digested by the aforementioned BamHI--NcoI was simultaneously cloned into the plasmid pWR13 that had been double-digested by BamHI and HindIII to obtain a fragment containing preS2 (120-146) and S(1 -226) the plasmid pWR/S2S of the fusion gene (see FIG. 1 ). The fusion clone was verified by DNA sequence determination, and it was confirmed that the reading frame after fusion was correct.

Example 2: PCR synthesis of preS1(21-47) epitope gene containing hepatocyte receptor binding site and construction of pWR/S2S fusion clone.

We selected the preS1 antigenic determinant of the binding site of hepatitis B virus hepatocyte receptor with adr subtype, the amino acid sequence number is 21-47, using the recombinant plasmid pADR-1 containing the preS1 gene as a template, and amplified by PCR A DNA fragment containing preS1 corresponding to positions 21-47 was identified. The 5' end primers used were:

5'CTTTCTGTTGTATACCCTCTGGG3'

AccI

The total length is 23 nucleotides, and the underlined part is an AccI restriction site designed and introduced at the 5' end of preS1. The primers used at the 3′ end are:

5′CCAGTGATAAGCTTAGGGGTGG3′

HindIII

The total length is 22 nucleotides, and the underlined part is a HindIII restriction site and a TAA termination code introduced at the 3′ of preS1. PCR and each method are the same as in Example 1. The PCR product was double-digested with AccI and HindIII for use. First, the fusion clone pWR/S2S containing preS2 (120-146) and S in Example 1 was opened by AccI and HindIII double enzymes (the 223rd position of the S gene contains an AccI restriction site), and inserted into the above PCR-synthesized A fragment containing preS1 and a TAA termination code was obtained to obtain a clone pWR/S2SS1 containing a triple fusion of preS2, S, and preS1 (see FIG. 2 ). Fusion clones were determined by DNA sequence, and the reading frame was proved to be correct for later use.

Example 3: Construction of recombinant vaccinia expression plasmid pGTP/S2SS1 containing preS2(120-146)+S(1-223)+preS1(21-47) fusion gene.

The recombinant plasmid pWR/S2SS1 was subjected to double enzymatic digestion with BamHI and HindIII to separate the triple fusion gene fragment containing preS2(120-146)+S(1-223)+preS1(21-47), which was directly inserted into the fragment opened by the same double enzymatic digestion Transition on plasmid pWR33 to obtain recombinant plasmid pWR33/S2SS1, then use BamHI and EcoRI double enzymatic digestion to separate the fusion gene fragment about 0.8kb in length, insert it into recombinant vaccinia expression plasmid pGJP-5 after the same double enzymatic digestion, and obtain the recombinant vaccinia expression plasmid pGJP-5 containing Cloning of preS2(120-146)+S(1-223)+preS1(21-47) triple fusion gene on recombinant vaccinia expression plasmid pGJP/S2SS1 (see Figure 3).

From the upstream, the recombinant plasmid is TK gene, vaccinia promoter p7.5, ATG code + preS2 (120-146) + ATG code + S (1-223) + preS1 (21-47) + TAA termination code, multiple cloning site, TK gene. The recombinant plasmid pGJP/S2SS1 also has an Apr drug resistance marker. It can be transformed in E. coli (eg TG-1), selected and amplified to prepare recombinant plasmid DNA. Escherichia Coli TG-1/pGJP/S2SS1 containing recombinant plasmid pGJP/S2SS1. Deposit mark CCTCC No.M96003.

Example 4: Screening of recombinant vaccinia virus containing preS2(120-146)+S(1-223)+preS1(21-47) fusion gene and identification of vS2SS1.

Recombinant vaccinia is produced by recombination of recombinant vaccinia expression plasmid pGJP/S2SS1 and Tiantan virus in CV-1 cells, and then by infecting TK-143 cells, the recombinant vaccinia plaques are selected in the presence of BUdR. Because the P7.5 promoter and fusion gene are inserted in the expression plasmid TK gene, TK has lost its activity, that is, TK-. After in vivo recombination, the recombinant vaccinia is also TK-, that is, in the presence of BUdR, only the recombined TK - To survive, after 2 times of plaque purification and positive expression of HBsAg, preS1 and preS2 antigens, the recombinant vaccinia virus vS2SS1 was selected.

The recombinant vaccinia virus vS2SS1 was identified by Southern blot at the DNA level. The vS2SS1 virus DNA was prepared, and the Tiantan strain virus DNA and the expression plasmid pGJP/S2SS1 containing the fusion gene were used as a control. Both were first digested with BamHI and EcoRI, electrophoresed, and then transferred to a nitrocellulose membrane. The S gene fragment was used as a probe for hybridization, and the results are shown in FIG. 4 . It is known that the recombinant expression plasmid pGJP/S2SS1 contains preS2(120-146)+ATG code+S(1-223)+preS1(21-47), and the fusion gene is about 0.8kb in length, while the recombinant vaccinia virus VS2SS1 was hydrolyzed by BamHI and EcoRI The fragment that can hybridize with the S gene is also a 0.8kb fragment, while the Tiantan strain control is negative, which shows that the fusion gene has been correctly inserted into the recombinant vaccinia virus DNA.

Example 5: Western blot analysis of recombinant vaccinia virus VS2SS1 expression product with preS2, S, preS1 triple antigenicity.

Recombinant vaccinia virus vS2SS1 and recombinant vaccinia virus vTH-2 containing only S gene were used as controls. After infecting Vero cells respectively, the culture medium was collected, and the expression products were analyzed by Western blot. After PAGE electrophoresis, transfer to nitrocellulose membrane. They were hybridized with anti-HBsAg monoclonal antibody (H166), anti-preS1 monoclonal antibody (125E11) and anti-preS2 monoclonal antibody (25-19). Using vTH-2 and vaccinia virus as controls, the results are shown in Figure 5.

Hybridization with S monoclonal antibody shows that vTH-2 expresses HBsAg 24KD and 27KD (glycosylated band) protein bands from the two columns in Figure 5A, while the protein of S2SS1 expressed by vS2SS1 is larger than S, showing 27KD, 29KD, 31KD and 33KD Proteins with different degrees of glycation (column A3 in Figure 5). If the anti-preS1 monoclonal antibody is used to hybridize, because there is no preS1 in vTH-2, it is negative (B2 column in Figure 5), and proteins with different degrees of glycation of 27KD, 29KD, 31KD and 33KD can also be seen in vS2SS1 (B3 in Figure 5 List). If the anti-preS2 monoclonal antibody is used for hybridization, it is also negative because there is no preS2 in vTH-2 (column C2 in Figure 5), while 31KD and 33KD protein bands can be seen in vS2SS1. Because the vS2SS1 gene structure contains two start codes starting from S2 and starting from S. Only the 31KD and 33KD of the product starting from S2 contain preS2 components and are positive for preS2 monoclonal antibody hybridization. If the product starts from S, at the 27KD and 29KD protein positions, because there is no preS2 component, it cannot hybridize with the preS2 monoclonal antibody and is negative. All vaccinia virus controls in the experiment were negative. This result clearly shows that the S2SS1 protein expressed by vS2SS1 has triple antigenicity of preS1 and preS2 in addition to HBsAg.

Example 6 The antigenicity and secretion of S2SS1 protein, the expression product of recombinant vaccinia virus vS2SS1.

Infect Vero cells with recombinant vaccinia virus vS2SS1, and use recombinant vaccinia virus vTH-2 containing only the S gene as a control at the same time. After infection, culture medium (extracellular m) and infected cell lysate (intracellular c). According to the above materials The expression levels of HBsAg, preS1 and preS2 antigens were determined by the ELISA method described in method g. It can be seen from Table 1 that the expression products of vS2SS1 can clearly detect the presence of HBsAg, preS1 and preS2 antigens in both culture medium and cell lysates, and all three antigens are secreted into the extracellular antigenic proteins respectively. It accounts for about 60% of the total amount of expressed antigenic protein. In the control vTH-2, there was no production of preS1 and preS2 antigens because only the S gene was present. At the same time, the S protein secreted by vTH-2 into the culture medium accounts for about 50% of the total antigenic protein expressed. It can be seen that compared with vTH-2, the S2SS1 protein expressing triple antigenicity has better secretion than the S protein.

Table 1. ELISA assay of recombinant vaccinia virus vS2SS1 expressing triple antigens: recombinant virus HBsAg preS1 preS2 vS2SS1 m c m c m c 1.044 0.581 0.870 0.692 0.311 0.231 vTH-2 1.000 1.068 0.034 0.034 0.013 0.027

m --- culture medium; c --- cell lysate; the value is OD490nm value.

Example 7 Using density ultracentrifugation and electron microscopy to observe the particle size analysis of S2SS1 protein expressed by recombinant vaccinia virus vS2SS1.

After infecting Vero cells with recombinant vaccinia virus vS2SS1 for 96 hours, the post-infection medium was collected and analyzed by cesium chloride density gradient ultracentrifugation. The antigenicity of HBsAg, preS1 and preS2 was tested according to the ELISA method described in Materials and Methods g. Determination. Firstly, the HBsAg antigenicity was determined, and an ELISA positive peak with a density of 1.22 g/ml could be found (see Figure 6). The antigenicity of preS1 and preS2 was also determined. Their ELISA positive peaks completely overlap with the former positive peaks, and they are also at the peak with a density of 1.22g/ml. This indicated that the expressed S2SS1 fusion protein had particles with a density of 1.22 g/ml and had triple antigenicity of HBsAg, preS1 and preS2. The antigenic protein contained in the peak was collected and observed under an electron microscope after negative staining, and S2SS1 antigenic particles with a diameter of about 22nm could be seen. The size, shape (22nm) and density (1.20/ml) of this antigen particle are not significantly different from the usual S antigen particle (Figure 7).

Claims (5)

1. one kind makes up with genetic engineering technique, contain the antigenic novel hepatitis B surface antigen of anterior surface antigen S1, S2 immunologic determinants and S simultaneously, and express to obtain to have preceding S1 by recombinant vaccinia system stably excreting, triple antigenic fusion particle of preceding S2 and S is characterized in that:

A. in the fusion gene of expressing, the pre S 1 antigen determinant that has comprised the liver cell binding site in the adr hypotype hepatitis B virus gene, the nucleotide sequence of corresponding 21-47 amino acids, also comprise the preS 2 antigen determinant that contains T cell and B cell epitope simultaneously, the nucleotide sequence of corresponding 120-146 amino acids, and the nucleotide sequence of corresponding 1-223 amino acids in the S surface antigen;

B. on the 1st amino acids position of 5 of S surface antigen gene ' end with the gene fusion of preS2 (120-146), simultaneously on 3 of the S surface antigen gene ' end the 223rd amino acids position with the gene fusion of preS1 (21-47);

C. the expression plasmid that uses in the vaccinia virus recombinant system of the fusion gene of Zu Jianing is pGJP-5/S2SS1, changes in the intestinal bacteria and deposits, and is labeled as CCTCC No.M96003;

D. vaccinia virus recombinant has triple fusions of hbsag gene and preS2 (120-146) epitopes gene and preS1 (21-47) epitopes gene, can stablely produce the fusion rotein S2SS1 with particle form with secretion property, this particle has triple antigenicities.

2. also comprise according to the gene fusion described in the claim 1 and contain hepatitis B surface S antigen gene 3 ' the 223rd of end coded amino acid and preS1 (21-47) fusion, or in the 1st of 5 ' coded amino acid and preS2 (120-146) fusion, (being the dual antigen of S2 and S or S and S1) and fusion (being triple antigens of S2, S and S1) simultaneously.

3. the hepatitis B surface S antigen gene that has according to claim 1, the fusion gene of anterior surface antigen S2 (120-146) and anterior surface antigen S1 (21-47) also can directly be used for the genetic immunization and the gene therapy of hepatitis B with dna form.

4. according to the fusion gene described in the claim 1, can be used for other outer expression systems of recombinant vaccinia expression system, as: mammal cell line system, Yeast system, fungi, bacterial system, insect viruses system etc.

5. fusion rotein S2SS1 according to claim 1 can be used as novel hepatitis B subunit particle vaccines, can more effectively be used for the prevention and the treatment of hepatitis B.

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