Study On Recombinant Human Antibody On Hepatitis B Virus Surface Antigen

Hepatitis B virus belong to Orthohepadnavirus of Hepadnaviridae,and HBV infection is a serious threat to human life and health, which can cause acute and chronic hepatitis,liver failure, cirrhosis, hepatocellular carcinoma and even death. About30million people worldwide have been infected with HBV, and nearly350million people with chronic HBV infection.in1992and2006, HBV infection seroepidemiological survey nationwide results show that, after HBV vaccinoprophylaxis included into the neonatal EPI, the HBsAg positive rate of the general population has decreased significantly, from9.75%in1992down to7.18%in2006, making China reduced to moderate endemic areas from high-endemic areas, but it still can be calculated that nearly93million people are HBV carriers, and among them20million people are chronic hepatitis B patients. The Prevalence Status Of Viral Hepatitis In China and Associated Issues Analytical report estimate China’s annual direct and indirect medical costs of chronic hepatitis B(including cirrhosis, liver cancer) is about600billion yuan, which Caused huge economic burden to patients and the country.Hepatitis B treatment has a long way to go, among the treatment drugs such as interferon, thymosin al, nucleoside analogues, peptide drugs and therapeutic vaccines and herbal medicine treatment, we have not yet found a drugs can be eradicated HBV. High titers of Hepaititis B immunoglobulin which is used in clinical and extracted from blood of HBsAb+healthy people can neutralize and clear the hepatitis B virus, so that the body will not be infected with the hepatitis B virus.Because of its high costs,limited source of blood and potential risk of pathogen infection,It makes the mass production of humanized antibodies possible after having screened high affinity specific monoclonal antibodies.based on this,The research of screening anti-HBsAg antibodies comtains three parts, as the following:1. Construction, panning and screening of human recombinant Fab antibody library to Hepatitis B surface antigen.the lymphocytes were isolated from the peripheral blood of five hepatitis B surface antibody positive (HBsAb+) volunteers after boostering immunization of domestic Hepatitis B vaccine, The total RNA was extracted from the human isolated lymphocytes and the cDNAs was synthesized with primer oligo-dT.The genes of Fd fragment and light and heavy chain of antibodies were amplified by a set of human IgG Fab antibody primers, eg.8pairs lambda light chain library primers,5pairs kappa light chain primers and8pairs heavy chain primers. The gel purified light chains and heavy chains were cloned into phagmid vector pComb3by restriction sites Sacl/Xbal and Xhol/SpeⅠ respectively, a phage antibody library with size3×108was constructed. Insertion rate of light and heavy chain inserts were nearly100%, fully meet the needs of our library screen.Successfully based on the Construction of phage antibody library, Helper phage VCSM13were used to packaged into Fab phage library, The library were panned and enriched by using commercialized and purified HBsAg with phage displaying. After third round of the panning,52human Fab clones were selected with reactivity to HBsAg by ELISA, Sequence analysis of52selected Fab clones revealed the presence of twenty unique clones, ELISA experiments show that these antibodies are specific for HBsAg, Competitive ELISA showed three different epitopes against HBsAg were indentified among20antibodies.2. Expression and identification of IgG antibodies in recombinant baculovirus/insect system and mammalian cell systems.On the Basis of successful expression of Fab antibody in XL1-Blue, we use mammalian transient expression systems and baculovirus-insect cell stable expression systems platform to achieve secretory whole antibody in eukaryotic cells. light chain and heavy chain Fd genes of the selected high titer Fab antibodies were cloned into IgG expression vector (pAc-L-FcR), and transfected into Sf9insect cell; at the same time, light chain and heavy chain Variable region genes were cloned into IgG expression vector (HL51-14),and transfected into293T mammalian cells. we detected the effect of transfection by direct immunofluorescence, then purified the supernatant that expressed from insect cells and mammalian cells, detected the antibody purity by SDS-PAGE, tested the function activity by ELISA.3. Affinity research of complete antibody against HBsAg.non-competitive ELISA and BIAcore results showed one antibody has a high affinity whose KD reach as much as10-9M, no statistical differences were found between mammal cell and insect cell expression system.In summary, we applied genetically engineered antibody preparation technology platform and phage display technology, and isolated the lymphocytes from the peripheral blood of five hepatitis B surface antibody positive (HBsAb+) volunteers after boostering immunization of domestic Hepatitis B vaccine to get Fab antibody gene, then we successfully constructed of phage antibody library, which had the size of3×108clones per ml, and its insertion rate of light and heavy chain inserts were nearly100%, fully meet the needs of our library screen. The library were panned and enriched by using commercialized and purified HBsAg with phage displaying. After the test of Competitive ELISA and sequence analysis three different epitopes against HBsAg were indentified among20unique sequence antibodies。After that we used mammalian transient expression systems and baculovirus-insect cell stable expression systems platform to achieve secretory whole antibody in eukaryotic cells. Light chain and heavy chain Fd genes of the selected high titer Fab antibodies were cloned into IgG expression vector (pAc-L-FcR), and transfected into Sf9insect cell; at the same time, light chain and heavy chain variable region genes were cloned into IgG expression vector (HL51-14),and transfected into293T mammalian cells. we detected the effect of transfection by direct immunofluorescence, then purified the supernatant that expressed from insect cells and mammalian cells, detected the antibody purity by SDS-PAGE, tested the function activity by ELISA. At last, we got7kinds of complete IgG antibodies. Results showed that antibodies from293T cells had higher titre against HBsAg. Then we detected the affinity of antibodies by non-competitive ELISA and BIAcore, Dissociation constants of antibodies by non-competitive ELISA from Sf9cells were1.06×10-8M、2.61×10-9M、2.50×10-10M, and from293T cells were1.42×10-10M、1.61×10-10M、1.47×10-10M、3.57×10-10M;Dissociation constants of antibodies by BIAcore from Sf9cells were1.06×10-8M、2.61×10-8M、2.50×10-10M, and from293T cells were1.42×10-8M、1.61×10-8M、1.47×10-8M、3.57×10-8M. Compared to non-competitive ELISA, BIAcore, with better and conservative persuasion, did not require labeled antibodies and could monitor the amount, speed, strength, specificity, and stability of antibody binding to antigen in the real time. we got IgG3Complete antibody of high affinity, if it can be used in Optimized proportion of antigen-antibody complex with hepatitis B surface antigen, we may get better treatment in urgent need of the next step the experimental results.