| Objectives: Hepatitis B virus is a very dangerous pathogen for human health. Till now, there is no effective therapeutic methods and solutions for chronic Hepatitis B virus infection. The poor development of the research on HBV infection mechanism is one of the most important reasons that make it difficult to develop the new anti-virus drugs and therapeutic methods. The research on HBV PreS1 region has significant meaning, since HBV PreS1 may play important role in HBV infection. However, the further study on HBV PreS1 has not been carried out because there is lack of specific monoclonal antibodies against this protein. In this study, we constructed the prokaryotic expression vectors to express HBV PreS1 in E.coli and to obtain purified PreS1 fusion protein. Then, the specific monoclonal antibodies (McAbs) against HBV PreS1 were prepared by hybridoma cell techniques, which served as the basement for further study.Methods:Firstly: PCR was deployed to amplify PreS1 gene segment containing Hindâ…¢and Xbaâ… endoenzyme sites. After double enzyme digestion, prokaryotic expression vectors pGST-MOLUC and PCR products were conjuncted by T4DNA ligase and converted into E.coli JM109 to construct and biopan the recombinant plasmids which were transformated into Eco-BL21(DE3) later. After IPGT induction, the fusion proteins were dealt with SDS-PAGE electrophoresis analysis and Western-blot detection. Then use glutathione-Sepharose 4B gelatum as filler to further purify the fusion proteins with affinity chromatograph.Secondly: Balb/c mice were immunized with PreS1 fusion protein and monoclonal antibodies against PreS1 were prepared with hybridoma method. The positive hybridomas secreting anti-PreS1 McAb were screened by means of ELISA. The antibody activity and specificity were analyzed by the indirect ELISA assay. The McAb immunoglobulin (Ig) subtype and the titer of the selected PreS1 McAb were determined. The specificity of monoclonal antibody was tested by Western blot analysis. The binding site of McAb were preliminarily determined by indirect ELISA test.Results:Firstly: the target gene segment, just the same size as expected, was amplified by PCR. After double enzyme digestion identification and sequence analysis, the recombinant plasmids were successfully cloned and named pGST–PreS1. And through successful transformation and induction, we got the expression molecular weight– 37kD- just the same as expected. And the SDS-PAGE showed that the expression level of fusion proteins were about 10% of total soluble proteins in lysate of expression bacteria. The Western-blot detection showed that inductive expressed fusion proteins can have specific bindings with GST single antibodies and Pres1 single antibodies. With the affinity chromatography of glutathione-Sepharose 4B, we obtained the fusion proteins with a purity of 90%.Secondly: from over thirty positive hybridoma which secreting monoclonal antibody to PreS1 fusion protein, we screening out a pair of hybridomas, named P1A1 and P2B3, Chromosome analysis revealed that the selected hybridoma was with the universal characteristics of the monoclonal hybridoma cells which secreted McAb, and the Ig subtype of P1A1 and P2B3 McAb is IgG2b , IgG3 subclass respectively. Balb/c mice were primed with pristine and then inoculated with well-grown hybridomas intraperitoneally. The induced ascites were proved containing anti-PreS1 monoclonal antibody with enzyme-linked immunoadsordent assay (ELISA) and their titer reached about 10-7; Eptitope recognized by P1A1 is different from that by P2B3; Western blot identification suggested that McAb could bind specially to the antigen.Conclusion:The PreS1 fusion proteins were obtained by recombinant DNA techniques and prokaryotic expression methods, and the specific monoclonal antibodies against PreS1 were prepared. The obtained monoclonal antibodies with high biological activity belong to the subtypes IgG2b and IgG3, respectively, which served as the foundation for further research on biological feature and function of PreS1 in HBV infection. |
