| Antibody is an important ingredient of human immune system, which could provide human body useful immune protection. With the development of medicine, man-made monoclonal antibodies are widely used in various domain of biomedical area. So far, the techniques used for preparing monoclonal antibodies include hybridoma technique, antibody library technique, antibody display technique, antibody screening technique and etc.Hybridoma technique appeared in the1970’s, which was the first technique used for prepare monoclonal antibodies in vitro. People could make antibodies with specific antigen affinity using hybridoma technique, which provide much progress to biomedical research. As the antibodies prepared using hybridoma technique have some mouse antibody portion, when they were used in human body, the human anti-mouse antibody (HAMA) responds could be caused, which was sometimes very dangerous. After the appearance of human-mouse chimeric antibody and re-shaping antibody, the risk of HAMA was decreased. But neither chimeric antibody nor reshaping antibody was fully human antibody. As scientists successfully make the antibody library technique and antibody display technique, the fully human antibodies could be prepared in vitro, by which could evade the HAMA response. Now, some antibody display and antibody screening techniques are widely used, such as phage display, yeast display, together with some novel techniques, such as E. coli display and mammalian display. These antibody display techniques all have some disadvantages. The most widely used phage display technique, as well as yeast display technique, could not display full-length antibody, but only successfully display some small antibodies, like fragment of antigen binding (Fab) or single chain variable fragment (scFv). After transfer to full-length antibody for producing in mammalian system, the specificity and affinity to antigen always changed, which also usually are not cost and/or time efficient. Although E. coli display technique could be used to display and screening full-length antibody, the antibody screened from E. coli display technique will not always be well produced in mammalian antibody producing system because of the different codons between E. coli and mammalian. So, the full-length antibody mammalian display system is needed. But the present full-length antibody mammalian display technique could only construct full-length antibody library with size of106, which was not large enough for screening of antibody with high specificity and high affinity.In this work, we firstly use the universal mammalian display vector pDGB-HC-TM to construct the large full-length antibody library. After digesting with restriction endonucleases BsmBI and SfiI respectively, we could pop-into pDGB-HC-TM the genes of antibody heavy chain variable domain or antibody light chain, then the gene library of antibody heavy chain and antibody light chain were constructed. By cotransfection of the two gene libraries, the host mammalian cell could assemble antibody heavy chain and antibody light chain, and display the full-length antibody on the cell surface. Using these strategies, we construct the big full-length antibody library with size of1.82×1011and diversity of8.71×1O10. Secondly, we used another mammalian display vector pDGB4to construct a four way ligation antibody library. The vector pDGB4contains dual mammalian expression cassettes for the one-step insertion of both heavy chain genes and light chain genes to display full-length bivalent antibodies on mammalian cell surfaces. After the four way ligation library was stably transfected into FCHO cell, a cell library which could stably display full-length antibodies was successfully constructed. We could detect that there were40.12%cells in the cell library could display full-length library. This cell library would be used for screening of specific antibody.Type B hepatitis is a cosmopolitan disease, which is caused by infection of hepatitis B virus (HBV). According to the data from2006, there were about100,000,000people in China who carried the hepatitis B virus surface antigen (HBsAg). The vertical transmission of HBV from pregnant to infant is one of the major routes of HBV infections. The use of hepatitis B immunoglobulin (HBIG) in pregnant is a helpful measure to protect fetus in uterus from infection of HBV. As the HBIG was produced from human blood, there would be some risks and the yield would not be well controlled. If anti-HBsAg antibody could be screened from antibody library, HBIG could be produced in vitro, which would be a more safe and controllable way to produce HBIGIn our research, we chose a healthy donor who was natural immuned by HBV. There was a high level of anti-HBsAg antibody in his peripheral blood. Use his peripheral blood lymphocyte, we isolated the antibody genes and constructed a HBV specific antibody library. After stably transfection of the antibody library into FCHO, we constructed a cell library display full-length antibody on cell surfaces. Coupled with high-flux fluorescence-activated cell sorting (FACS), we screened out eight cell clones which could bind HBsAg. After sequencing the antibody genes in the eight cell clones, we make sure that there were three unique antibodies among the eight cell clones, and the three unique antibodies had different affinity to HBsAg.In a word, we successfully used the full-length antibody mammalian display technique to screen specific antibody, and the three unique antibodies we screened would be useful in producing HBIG in vitro. |
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