Insulin resistance reduces sensitivity to Cis-platinum and promotes adhesion, migration and invasion in HepG2 cells.

The liver is normally the major site of glucose metabolism in intact organisms and the most important target organ for the action of insulin. It has been widely accepted that insulin resistance (IR) is closely associated with postoperative recurrence of hepatocellular carcinoma (HCC). However, the relationship between IR and drug resistance in liver cancer cells is unclear. In the present study, IR was induced in HepG2 cells via incubation with a high concentration of insulin. Once the insulin-resistant cell line was established, the instability of HepG2/ IR cells was further tested via incubation in insulin-free medium for another 72h. Afterwards, the biological effects of insulin resistance on adhesion, migration, invasion and sensitivity to cis-platinum (DDP) of cells were determined. The results indicated that glucose consumption was reduced in insulin-resistant cells. In addition, the expression of the insulin receptor and glucose transportor-2 was downregulated. Furthermore, HepG2/IR cells displayed markedly enhanced adhesion, migration, and invasion. Most importantly, these cells exhibited a lower sensitivity to DDP. By contrast, HepG2/IR cells exhibited decreased adhesion and invasion after treatment with the insulin sensitizer pioglitazone hydrochloride. The results suggest that IR is closely related to drug resistance as well as adhesion, migration, and invasion in HepG2 cells. These findings may help explain the clinical observation of limited efficacy for chemotherapy on a background of IR, which promotes the invasion and migration of cancer cells.

Coimmunization with IL-15 plasmid enhances the longevity of CD8 T cells induced by DNA encoding hepatitis B virus core antigen.

AIM: To test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for improving the immune responses induced by hepatitis B virus core gene DNA vaccine. METHODS: We used RT-PCR based strategies to develop IL-15 expression constructs. We first confirmed that the gene could be expressed in Escherichia coli due to the poor expression of IL-15. Then the bioactivity of IL-15 plasmid expression product was identified by CTLL-2 proliferation assay. One hundred micrograms of DNA from each of the IL-15 eukaryotic expressed plasmid and the recombinant plasmid harboring DNA encoding the 144 amino acids of the N-terminus of HBV core gene (abbreviated pHBc144) was used to co-immunize C57 BL/6 mice. The titer of anti-HBcIgG was detected by ELISA and the antigen-specific CD8(+) T cells (CD8(+)IFN-gamma(+) T cells) were detected by intracellular cytokine staining at different time points. RESULTS: After co-immunization by pIL-15 and pHBc144 DNA vaccine the antigen-specific CD8(+) cells of mice increased gradually, the first peak of immune response appeared 14 d later, then the number of antigen-specific CD8(+) Ts cells decreased gradually and maintained at a steady level in 3 mo. After boosting, the number of antigen-specific CD8(+) T cells reached the second peak 10 d later with a double of the 1st peak, then the number of antigen-specific CD8(+) T cells decreased slowly. IL-15 as a gene adjuvant had no significant effect on humoral immune responses induced by hepatitis B virus core gene DNA vaccine, but increased the memory antigen-specific CD8(+) T cells induced by hepatitis B virus core gene DNA vaccine. CONCLUSION: DNA vaccine constructed by HBc Ag 1-144 amino acid induces effective cell immunity, and cytokine plasmid-delivered IL-15 enhances the longevity of CD8(+) T cells.

Identification of the immunogenic domains in HBsAg preS1 region using overlapping preS1 fragment fusion proteins.

AIM: The incorporation of hepatitis B virus (HBV) preS1 region into epitope-based vaccines against HBV has been accepted widely, but the incorporate site and size of preS1 sequence is controversial. Therefore our purpose was to further investigate its immunogenic domains for the epitope-based hepatitis B vaccine design. METHODS: Eight GST fusion proteins containing overlapping preS1 fragments in preS1 (21-119) region were expressed in E.coli. Using these purified fusion proteins, the immunogenic domains in preS1 region were identified in detail in mice and humans by Western blot analysis and ELISA. RESULTS: The results in mice showed that the immu-nogenic domains mainly existed in preS1 (21-59) and preS1 (95-109). Similarly, these fragments had strong immunogenicity in humans; whereas the other parts except for preS1 (60-70) also had some immunogenicity. More importantly, a major immunogenic domain, preS1 (34-59), which has much stronger immunogenicity, was identified. Additionally, the antibodies against some preS1 fragments, especially preS1 (34-59), were speculated to be virus-neutralizing. CONCLUSION: Eight GST fusion proteins containing overlapping preS1 fragments were prepared successfully. They were used for the study on the immunogenic dom-ains in preS1 (21-119) region. The preS1 (34-59) fragments were the major immunogenic domains in the preS1 region, and the antibodies against these fragments were speculated to be virus-neutralizing. Therefore, the incorporation of preS1 (34-59) fragments into epitope-based HBV vaccines may be efficient for enhancement of immune response. Additionally, the results also imply that there are more complex immune responses to preS1 region and more abundant immunogenic domains in humans.

Purification and application of C-terminally truncated hepatitis C virus E1 proteins expressed in Escherichia coli.

AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli (E. coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally truncated E1 fragments were expressed in E. coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chromatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot. RESULTS: Full-length E1 protein proved difficult to express in E. coli. C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni(2+)-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 glycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization. CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E. coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.

Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells.

AIM: To explore the role of hepatitis C virus (HCV) envelope protein 2 (E2) in the induction of apoptosis. METHODS: A carboxyterminal truncated E2 (E2-661) was transiently expressed in several cultured mammalian cell lines or stably expressed in Chinese hamster ovary (CHO) cell line. Cell proliferation was assessed by 3H thymidine uptake. Apoptosis was examined by Hoechst 33258 staining, flow cytometry and DNA fragmentation analysis. RESULTS: Reduced proliferation was readily observed in the E2-661 expressing cells. These cells manifested the typical features of apoptosis, including cell shrinkage, chromatin condensation and hypodiploid genomic DNA content. Similar apoptotic cell death was observed in an E2-661 stably expressing cell line. CONCLUSION: HCV E2 can induce apoptosis in cultured mammalian cells.

A candidate DNA vaccine elicits HCV specific humoral and cellular immune responses.

AIM: To investigate the immunogenicity of candidate DNA vaccine against hepatitis C virus (HCV) delivered by two plasmids expressing HCV envelope protein 1 (E1) and envelope protein 2 (E2) antigens respectively and to study the effect of CpG adjuvant on this candidate vaccine. METHODS: Recombinant plasmids expressing HCV E1 and E2 antigens respectively were used to simultaneously inoculate mice with or without CpG adjuvant. Antisera were then collected and titers of anti-HCV antibodies were analyzed by ELISA. One month after the last injection, animals were sacrificed to prepare single-cell suspension of splenocytes. These cells were subjected to HCV antigen specific proliferation assays and cytokine secretion assays to evaluate the cellular immune responses of the vaccinated animals. RESULTS: Antibody responses to HCV E1 and E2 antigens were detected in vaccinated animals. Animals receiving CpG adjuvant had slightly lower titers of anti-HCV antibodies in the sera, while the splenocytes from these animals showed higher HCV-antigen specific proliferation. Analysis of cytokine secretion from the splenocytes was consistent with the above results. While no antigen-specific IL-4 secretion was detected for all vaccinated animals, HCV antigen-specific INF-gamma secretion was detected for the splenocytes of vaccinated animals. CpG adjuvant enhanced the secretion of INF-gamma but did not change the profile of IL-4 secretion. CONCLUSION: Vaccination of mice with plasmids encoding HCV E1 and E2 antigens induces humoral and cellular immune responses. CpG adjuvant significantly enhances the cellular immune response.

Expression of hepatitis C virus E2 ectodomain in E. coli and its application in the detection of anti-E2 antibodies in human sera.

The second envelope glycoprotein (E2) of hepatitis C virus has been shown to bind human target cells and has become a major target for the development of anti-HCV vaccines. Anti-E2 antibodies have been suggested to be of clinical significance in diagnosis, treatment and prognosis of hepatitis C. However, large-scale expression and purification of E2 proteins in mammalian cells is difficult. As an alternative, E2 fragment (aa 385 C730) with a four-amino-acid mutation (aa 568 C571 PCNI to RVTS) was expressed as hexa-histidine-tagged full length protein [E2N730(m)] in E. coli and purified to over 85% purity. Purified E2N730(m) was specifically recognized by homologous hepatitis C patient serum in Western blot, suggesting that it displayed E2-specific antigenicity. Rabbit antiserum raised against E2N730(m) recognized E2 glycoproteins expressed in mammalian cells in Western blot. Purified E2N730(m) was used to detect anti-E2 antibodies in human sera and showed better specificity and sensitivity than previously reported C-terminally truncated E2 fragment (aa 385 C565). Association between anti-E2 antibodies in patient sera and HCV RNA status was also demonstrated using this E. coli-derived protein. E2N730(m) might serve as an inexpensive alternative to mammalian cell-expressed E2 proteins in clinical and research applications.

Expression and characterization of hepatitis C Virus E2 glycoprotein fused to hepatitis B virus preS1(21-47) fragment in CHO cells.

To stably express hepatitis C virus (HCV) E2 glycoprotein in CHO cells and facilitate the detection and purification of the expression products, the gene fragment encoding N-terminal 277 amino acids of this protein was fused to the fragment encoding hepatitis B virus (HBV) preS1(21-47) region and inserted into a secretion vector pSecTagB. CHO cells transfected with the recombinant plasmid carrying fusion gene were selected under growth pressure of Zeocin. Secreted fusion products and its cell-associated counterpart were detected by Western blot using E2 specific or preS1 specific antibodies. Glycans carried by the expression products were analyzed with glycan-type specific glycosidases. Most of the cell-associated E2 were found to be high-mannose-type glycosylated, while the secreted E2 proteins were found to be mostly complex-type glycosylated, suggesting further modification in Golgi apparatus upon secretion. Primary studies showed that the fusion antigen could be specifically bind to and elute from anti-preS1 antibody coupled Sepharose resin, suggesting that large-scale preparation of the fusion antigen is feasible with an immunoaffinity resin. This work will contribute to the further study of immunological properties of HCV E2 glycoprotein and also to the study of recombinant HBV/HCV vaccine.

DNA immunization with recombinant HCV E2 expression plasmids.

DNA-based immunization, the delivery of plasmid DNA by direct inoculation, is a newly developed method of vaccination. Besides of many advantages, DNA immunization was demonstrated to generate weaker antibody and CTL responses than did protein and live attenuated vaccines, respectively. To circumvent this shortage, several methods were tested such as using different vector, changing injection mode, and co-expressing cytokines, etc. The studies showed that many factors could greatly affect the immune responses to DNA vaccine. The aim of this study is to compare different vectors for the presentation of the HCV E2 to generate immune responses by using DNA-based immunization. Four expression plasmids, with different promoter types and with or without signal sequences were constructed, which encode C-terminally truncated E2 (384-660) with HBV preS1 21-47 tag fused to its N-termini. Transient expression in HeLa cells showed that only recombinant plasmids with signal sequence could be expressed and properly processed, and the product could be secreted into medium. Protein expression level is slightly higher in plasmids with CMV promoter than with EF1alpha promoter. After immunization of C57BL/6 mice, all the recombinant constructs could elicit both anti-preS1 and anti-E2 antibodies. But only pCMV Sec-S1E2t660 with both CMV promoter and signal sequence could induce high-level and long-lasting antibody, showing that it is a good vaccine candidate. Possible reasons for the different immune responses to these constructs were discussed.

Full-length core sequence dependent complex-type glycosylation of hepatitis C virus E2 glycoprotein.

AIM: To study HCV polyprotein processing is important for the understanding of the natural history of HCV and the design of vaccines against HCV. The purpose of this study is to investigate the affection of context sequences on hepatitis C virus (HCV) E2 processing. METHODS: HCV genes of different lengths were expressed and compared in vaccinia virus/T7 system with homologous patient serum S94 and mouse anti-serum M( E2116) raised against E.coli -derived E2 peptide, respectively. Deglycosylation analysis and GNA ( Galanthus nivalus ) lectin binding assay were performed to study the post-translational processing of the expressed products. RESULTS: E2 glycoproteins with different molecular weights (-75 kDa and -60 kDa) were detected using S94 and M( E2116), respectively. Deglycosylation analysis showed that this difference was mainly due to different glycosylation. Endo H resistance and its failure to bind to GNA lectin demonstrated that the higher molecular weight form (75 kDa) of E2 was complex-type glycosylated, which was readily recognized by homologous patient serum S94. Expression of complex-type glycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected in constructs encoding full-length core sequences. CONCLUSION: The upstream conserved full-length core coding sequence was required for the production of E2 glycoproteins carrying complex-type N-glycans which reacted strongly with homologous patient serum and therefore possibly represented more mature forms of E2. As complex-type N-glycans indicated modification by Golgi enzymes, the results suggest that the presence of full-length core might be critical for E1/E2 complex to leave ER. Our data may contribute to a better understanding of the processing of HCV structural proteins as well as HCV morphogenesis.

DNA immunization with fusion genes encoding different regions of hepatitis C virus E2 fused to the gene for hepatitis B surface antigen elicits immune responses to both HCV and HBV.

AIM: Both Hepatitis B virus (HBV) and Hepatitis C virus (HCV) are major causative agents of transfusion-associated and community-acquired hepatitis worldwide. Development of a HCV vaccine as well as more effective HBV vaccines is an urgent task. DNA immunization provides a promising approach to elicit protective humoral and cellular immune responses against viral infection. The aim of this study is to achieve immune responses against both HCV and HBV by DNA immunization with fusion constructs comprising various HCV E2 gene fragments fused to HBsAg gene of HBV. METHODS: C57BL/6 mice were immunized with plasmid DNA expressing five fragments of HCV E2 fused to the gene for HBsAg respectively. After one primary and one boosting immunizations, antibodies against HCV E2 and HBsAg were tested and subtyped in ELISA. Splenic cytokine expression of IFN-gamma and IL-10 was analyzed using an RT-PCR assay. Post-immune mouse antisera also were tested for their ability to capture HCV viruses in the serum of a hepatitis C patient in vitro. RESULTS: After immunization, antibodies against both HBsAg and HCV E2 were detected in mouse sera, with IgG2a being the dominant immunoglobulin sub-class. High-level expression of INF-gamma was detected in cultured splenic cells. Mouse antisera against three of the five fusion constructs were able to capture HCV viruses in an in vitro assay. CONCLUSION: The results indicate that these fusion constructs could efficiently elicit humoral and Th1 dominant cellular immune responses against both HBV S and HCV E2 antigens in DNA-immunized mice. They thus could serve as candidates for a bivalent vaccine against HBV and HCV infection. In addition, the capacity of mouse antisera against three of the five fusion constructs to capture HCV viruses in vitro suggested that neutralizing epitopes may be present in other regions of E2 besides the hypervariable region 1.

Detection of anti-preS1 antibodies for recovery of hepatitis B patients by immunoassay.

AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B. METHODS: The expression plasmid pET-28a-preS1 was constructed, and a large quantity of preS1(21-119aa) fragment of the large HBsAg protein was obtained. The preS1 fragment purified by Ni(2+)-IDA affinity chromatography was used as coated antigen to establish the indirect ELISA based on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. For follow-up study, serial sera were collected during the clinical course of 21 HBV-infected patients and anti-preS1 antibodies, preS1 antigen, HBV-DNA and other serological HBV markers were analyzed. RESULTS: preS1(21-119aa) fragment was highly expressed from the plasmid pET-28a-preS1 in a soluble form in E.Coli (30mg.L(-1)), and easily purified to high purity over 90% by one step of Ni(2+)-IDA-sepharose 6B affinity chromatography. The purity and antigenicity of the purified preS1(21-119aa) protein was determined by 150g.L(-1) SDS-PAGE, Western blot and a direct ELISA. Recombinant preS1(21-119aa) protein was successfully applied in the immunoassay which could sensitively detect the anti-preS1 antibodies in serum specimens of acute or chronic hepatitis B patients. Results showed that more than half of 19 acute hepatitis B patients produced anti-preS1 antibodies during recovery of the disease, however, the response was only found in a few of chronic patients. In the clinical follow-up study of 11 patients with anti-preS1 positive serological profile, HBsAg and HBV-DNA clearance occurred in 6 of 10 acute hepatitis B patients in 5-6 months, and seroconversion of HBeAg and disappearance of HBV-DNA occurred in 1 chronic patients treated with lavumidine, a antiviral agent. CONCLUSION: The high-purity preS1(21-119aa) coated antigen was successfully prepared by gene expression and affinity chromatography. Using this antigen, a conveniently detective system of anti-preS1 antibodies in sera was established. Preliminarily clinical trial the occurrence of anti-preS1 antibodies in acute hepatitis B patients suggests the clearance of HBV from serum in a short-term time, and anti-preS1 positive in chronic patients means health improvement or recovery from the disease.

Development of the diagnostic immunoassay to detect anti-PreS1(21-47aa) antibody--a marker suggesting the health improvement of hepatitis B patients.

BACKGROUND: A new immunoassay has been developed for the detection of the anti-PreS1(21-47aa) antibody in sera of hepatitis B virus (HBV)-infected patients. Anti-PreS1(21-47aa) antibody involves virus neutralization and is a new marker for diagnosing acute and chronic B hepatitis. METHODS: The expression plasmids pGEXS I and pGEXS II, which expressed glutathione S-transferase (GST) fusion proteins containing a copy of PreS1(21-47aa) peptide and two orderly joined copies of PreS1(21-47aa) peptide, were constructed. The soluble expression products were purified by affinity chromatography. RESULTS: The two PreS1(21-47aa) fusion proteins were both successfully applied in the immunoassay based on biotin-protein A and streptavidin-HRP, and could detect the anti-PreS1(21-47aa) antibody with high sensitivity in sera from hepatitis B patients. The anti-PreS1(21-27aa) antibody was detected during the recovery phase of acute hepatitis B patients, but it was found only in few of the chronic carriers by the established conventional system. CONCLUSIONS: The follow-up study suggested that the presence of the anti-PreS1(21-27aa) antibody correlated well with the recovery of patients from hepatitis and the improvement in health.

High Frequency of Homologous Recombination in the Genome of Modified Vaccinia Virus Ankara Strain (MVA).

MVA is a genetically modified vaccinia virus strain, which is replication defective and extremely safe for research and clinical use. To further improve the safety of MVA and achieve more efficient selection of recombinant constructs, a transient selection marker system has been developed, which contains vaccinia virus k1l gene flanked by two identical fragments. In this report, four recombinant MVAs were constructed with this system, and homologous recombination of recombinant MVAs was studied during the construction procedure. The results showed that the recombination frequency of these rMVAs was significantly high, though lower than that observed in other vaccinia virus strains. The k1l free rMVAs could be conveniently obtained after 3 or 4 blind passages. Our data indicated that recombinant MVA with a transient selection marker system was safe for use as live vaccine and gene therapy vector for human. In addition, blind passages could enhance the efficiency in isolation of k1l free recombinants

Expression, Purification and Preliminary Clinical Use of Recombinant HBsAg GST-PreS1(21--47 aa) Fusion Proteins.

Expression plasmids pGEXSI and pGEXSII containing one copy and two orderly joined copies of PreS1(21--47 aa) DNA fragment, respectively, were constructed. GST-PreS1(21--47 aa) and GST-2xPreS1(21--47 aa) fusion proteins were highly expressed in E.Coli TG1, induced by IPTG. The expression level of GST-PreS1(21--47 aa) was about 30% of total soluble proteins in the lysate of expression bacteria, and GST-2xPreS1(21--47 aa) was about 15% of total soluble proteins, asestimated by SDS-PAGE. 50 mg GST-PreS1(21--47 aa) or 20 mg GST-2xPreS1(21--47 aa) with purity over 90% was obtained, respectively, from 1 L culture by using affinity chromatography of glutathione-Sepharose 4B. Direct ELISA results showed that antigenicity of GST-2xPreS1(21--47 aa) was better than GST-PreS1(21--47 aa) and synthetic peptide. Using GST-2xPreS1(21--47 aa) as coated antigen, a sensitive indirect ELISA for detection of anti-PreS1(21--47 aa) antibody, based on protein A-biotin and streptavidin-HRP, was established. The results from 99 sera samples of hepatitis B patients showed that anti-PreS1(21--47 aa) antibody was detected in nearly half of acute hepatitis B patients during recovery, but it was detected only in a few chronic hepatitis patients. Clinical follow-up study suggested that appearance of anti-PreS1(21--47 aa) was related to the course of the disease and recovery of patients. Detection system established in the study is promising for clinical application.

Purification and Characterization of Recombinant Hepatitis B Virus Surface Antigen SS1 Expressed in Pichia pastoris.

The purification of recombinant hepatitis B surface antigen, SS1 protein, expressed in Pichia pastoris, and the investigation of its physiochemical characters and immunogenicity were described here. Employing McAb immunoaffinity chromatography, this protein was purified to purity of 95%. The results of ELISA and Western blotting showed good antigenicity of this purified SS1 protein. CsCl gradient centrifugation and electron microscopy assay proved that this purified protein could be assembled into particles similar to the HBV subviral particles. Strong antibody responses against both the HBs and PreS1 epitopes were induced in BALB/c mice immunized with this purified protein. The simultaneous injection of a CpG adjuvant induced a Th1-like immune response against both the HBs and PreS1 epitopes.

Expression of the Recombinant Hepatitis B Virus Surface Antigen Carrying PreS Epitopes in Pichia pastoris.

Many studies have suggested that hepatitis B surface antigen(HBsAg) including PreS sequences could be an ideal candidate for highly effective hepatitis B virus vaccine. Modified surface antigens S1S, SS1 and S2S which carried PreS epitopes, were expressed in Pichia pastoris. The characterization of antigenicity and particle assembly demonstrated that the expression products could be assembled into particles which presented S, PreS1 or PreS2 antigenicity, respectively. The expression was more efficient than that in Saccharomyces cerevisiae.

Fusion Expression, Immunogenicity and Applications of C-terminally Truncated HCV Core Proteins.

Clones expressing full-length(aa 1-191) or C-terminally truncated forms (aa 1-69 and aa 1-40) of hepatitis C virus core(HCc) protein fused to the C-terminal of glutathione S-transferase(GST) were constructed and their expressions in different strains of Escherichia coli were compared. The expressed proteins were soluble. ELISA and Western blot analyses showed that GSTC191 was poorly expressed and had poor stability, whereas GSTC69 and GSTC40 were stable and could be purified to 90% purity in a single step on glutathione-Sepharose 4B. Mice immunized with these purified proteins produced high-titre antibodies. As an application, purified GSTC69 and GSTC40 were used in a preliminary assay of anti-HCc antibodies in patients'sera, and results showed that they had high specificity in the assay.

The Fusion Expression of HBV preS Epitopes and the Core Antigen.

The DNA fragments encoding the preS epitopes of HBV surface antigen were fused to the HBc gene and expressed in E. coli under the control of the tac promoter. The products were analyzed by ELISA and Western blotting, which confirmed that the hybrid proteins were expressed as expected. Analysis by electron microscopy and CsCl density gradient ultracentrifugation revealed that all fusion proteins were able to form particles, only with a slightly lower density than the native multimeric HBc. Partially purified fusion particles were then used as immunogen to Balb/c mice and high titer antibody against the preS1(21-47) epitope was observed, which demonstrated that the immunogenictiy of preS1 (21-47) could be greatly improved when fused in the el loop in HBc protein.

Affinity Purification of Hepatitis B Virus Surface Antigen Containing PreS1 Region.

The large protein of hepatitis B virus surface antigen (LHBs) contained an attachment site of HBV to liver cells and the antibodies to preS1 were virus-neutralizing. Therefore, vaccines containing preS1 would be more protective. However, One of the key problems in the preparation of gene-expressed proteins was the purification of the products. We have established a method of immuno-affinity chromatography with the anti-HBsAg-preS1 monoclonal antibody. Using this technique, We have successfully purified gene-expressed fusion protein of hepatitis B virus surface antigen containing preS1 (21-47). Results showed that this method was simple, effective and specific as compared to other methods, and might be useful in the future.