The Cloning And Expression Of Human Papillomavirus Type 16 E7 Gene In Escherichia Coli

HPV (human papillomavirus, HPV) is double-stranded DNA virus,approximately 7900 bp in length. HPV infections occur on skin or mucosal surface of the basal epithelial cells via direct contact. Human beings are the only host of HPV. Cervical HPV infection is the main causes of cervical cancer, HPV-16 and HPV-18 account for 60-70% of all cervical cancer cases worldwide. The mechanism in which HPV causes cervical cancer is rather complicated. Integration of viral DNA into host chromosomes and E6, E7 protein production coded by HPV are closely associated with formation of tumors. In summary, HPV E6, E7 genes, P53 and pRb genes are all involved in development of cervical cancer. In this study, we construct a prokaryotic expressing vector harboring HPV-16 E7 gene and this fundamental work make therapeutic vaccine applicable for cervical cancer cases. .Objective: To construct prokaryotic expressing vector harboring human papillomavirus-16 E7 gene and to evaluate E7 protein expressing.Method: (1) Polymerase chain reaction (PCR) and cloning: HPV-16 E7 fragment was synthesized, E7 fragment was amplified using polymerase chain reaction. The target gene was cloned into pMD18-T cloning vector. (2) Then the fragment was sequenced and inserted into expression vector pET-28a(+)and pGEX-4T-1, and the recombinant expressing vector pET-28a(+)-HPV16E7 and pGEX-4T-1-HPV16E7 was transformed into E. coli, Rosetta(DE3)and BL21(λDE3). (3) E7 protein expression: prokaryotic expression vector, the pET-28a (+)-HPV16E7 and the pGEX-4T-1-HPV16E7 in E. coli were cultured at 37℃overnight. The next day, expressing vectors in E. coli were treated with IPTG. SDS-PAGE electrophoresis was carried out to observe the optimal conditions of E7 protein expressing. (4) The amino acid sequence of E7 was performed using NCBI and bioinformatics analysis.Results: (1) A synthetic DNA fragment HPV16E7 was obtained with OD260/OD280 in 1.8-2.0 by UV spectrophotometer, it is consistent with PCR requirement. (2) HPV16E7 gene was successfully amplified, 297bp long and subsequently cloned into pMD18-T. (3) Prokaryotic expressing vector pET-28a (+)-HPV16E7 harboring human papillomavirus-16 E7 gene can't expressed aimed protein, in contrast to it, prokaryotic expression vector, pGEX-4T-1-HPV16E7 in E. coli may express E7 protein after IPTG treatment.(5) Bioinformatics analysis and forecasting results show that the number of amino acids is 98 and most of them consists of Leonine.Conclusions: (1) HPVl6 E7 gene was successfully amplified by PCR based on synthetic HPV16E7 sequence. (2) Prokaryotic expressing vector, pGEX-4T-1-HPV16E7, can express E7 fusion protein. (3) The optimal conditions of E7 fusion protein expressed by pGEX-4T-1-HPV-16E7 show that the high level of E7 protein can be acquired at 25℃and at the concentration of 0.4umol/L of IPTG. (4) Bioinformatics reveals E7 gene containing 98 amino acids.