Preparation And Identification Of Monoclonal Antibodies Against PreS2 Of Hepatitis B Virus

Objective:Previous studies have show that the protein of HBV pre S2 have the receptor of Polymenized Human Serum Albumin (PHSA), which could mediat the DANE partical's absorption into the surface of the hepatocytes and made important role in the HBV attacking hepatocytes. The HBV pre S2 protein contains several specific T,B lymphocytes binding site that could neutralizing some antibodies and induce the protective immunity. In this study, we are going to construct the prokaryotic expression vector with HBV PreS2 genes, express it highly in E.coli to get purified PreS2 fusion protein and then identify the protein's characterization at the first. Then, we are going to prepare monoclonal antibody (mAb) to PreS2 of hepatitis B virus by hybridoma technique. It is potentially useful to study the biologic function of PreS2 and further explore the role of PreS2 in Hepatitis B virus infection. Moreover, it provides us new insights into the preventative and clinical therapy of Hepatitis B virus.Methods:Firstly: PCR was deployed to amplify PreS2 gene segment containing Bgl II and SalⅠendoenzyme sites. After double enzyme digestion, prokaryotic expression vectors pGST-MOLUC and PCR products were conjuncted by T4DNA ligate 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 PreS2 fusion protein and monoclonal antibodies against PreS2 were prepared with hybridoma method. The positive hybridomas secreting anti-PreS2 mAb were screened by means of ELISA. The antibody activity and specificity were analyzed by the indirect ELISA assay. The mAb immunoglobulin (Ig) subtype and the titer of the selected PreS2 mAb were determined. The specificity of monoclonal antibody was tested by Western blot analysis. The binding site of mAb 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–PreS2. And through successful transformation and induction, we got the expression molecular weight– 33kD- 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 PreS2 fusion protein, we screening out a pair of hybridomas, named 7A61 and 5E73, Chromosome analysis revealed that the selected hybridoma was with the universal characteristics of the monoclonal hybridoma cells which secreted mAb, and the Ig subtype of 7A61 and 5E73 mAb is IgG1 ,.Balb/c mice were primed with pristine and then inoculated with well-grown hybridomas intraperitoneally. The induced ascites were proved containing anti-PreS2 monoclonal antibody with enzyme-linked immunoadsordent assay (ELISA) and their titer reached about 10-7; Eptitope recognized by 7A61 is different from that by 5E73; Western blot identification suggested that mAb could bind specially to the antigen.Conclusion:Through the above experiments, we obtained the inhibitory GST-PreS2 fusion protein and purified the target protein with high-purity and the correct molecular size. Two monoclonal antibodies against PreS2 protein were obtained and characterized finally. The results provide concrete experimental data for studying the biologic functions of PreS2 and further exploring the role of PreS2 in Hepatitis B virus infection. Moreover, it provides us new insights into the preventative and clinical therapy of Hepatitis B virus.