Preparation Of Type HPV16 E7 Protein Monoclonal Antibody

[Objectives] Human Papillomavirus (HPV) is a kind of DNA virus widely distributed in nature, which has a high infection rate in population. Infection rate HPV is the most important cause for tumors, including a variety of digestive tract tumors. After the genome of HPV16 integrated into celluar, HPV16 E7 protein can be persistently expressed with high level in it. HPV16 E7 protein can bind effectively with Rb, which is one of the most important tumor suppressor proteins for cell. Once at the time Rb is bound with HPV16 E7, it will finally enhance expressions of many proteins which are not expressed at normal. All of these are the important reasons which resulting in the cell being a condition named malignant transformation. So it is a popular view looking HPV16 E7 as a tumor specific antigen (TSA) for HPV16 related cancer and it is also confirmed as an ideal target for the immunotheraoy on HPV16 related cancers. Up to date, PCR is a main weapon used to find HPV infection, including HPV16. But as we know, PCR is depended on the equipment and it must be performed under a relatively separated and clean environment. Otherwise it will give us a lot of false positive information. Comparison with PCR, immunolocal method is more conwenient. The HPV 16 E7 monoclonal antibody was prepared to study the role nearly warning of cancer and develop therapeutic vaccines.[Methods] The plasmids constructed with PGEX4T-1-HPV16 E7 were transformed into E. coli BL21 strain. Expression of the E7 was induced by IPTG and confirmed by Western Blot, and its relative molecular massof the recombinant HPV16 E7 fusion protein was approximately 37kD. The fusion protein was purified by metal-ligand affinity chromatography, which should be used as diagnostic reagent of HPV16. Spleen cells from immunized Balb/c mice were fused with SP2/0 cells by a routing method, and the valence of antibody was evaluated by dot-spot ELISA. A series of the expansion, isolation and freezing of clones were carried out, and finally the ascites fluid which contained monoclonal antibodies was produced.[Results]1. After the recombinant vector was transformed into E.coli BL21, HPV16 E7 fusion protein was expressed induced by IPTG. Its relative molecular mass was approximately 37kD2. A series of the expansion, isolation and freezing of clones were carried out, and finally the ascites fluid which contained monoclonal antibodies was produced.Ascites production is between about 3～10ml and the titer is 10-6.3. The HPV16 E7 monoclonal antibody could be stained in a variety of cancers tissue nucleus.[Conclusion] The HPV16 E7 protein antibody is a specific McAb. This research has laid the foundation for further study of HPV16 biology function,detection of HPV16 E7,and immunology therapy.