The Preparation Of Hpv58e7 Protein By GST-tagged Prokaryotic Expression System

Background and aim: Cervical cancer is one of the major health issues in women of reproductive age, especially in developing counties. It has been confirmed that infection of high risk type of human papillomavirus is the key initiator. epidermiologically, The prevalence of high-risk HPV types in cervical cancers varies among different geographic areas. The most prevalent high-risk genital HPV types worldwide in cervical cancer are HPV16 and HPV18; it has been reported thatThe genotype 58 has been found in Asian populations including china prevalently.Two major viral oncogenes, E6 and E7 proteins of high risk HPV types, which possess the transforming property, play a key role in HPV-related carcinogenesis. Interaction of E6 oncoprotein with p53 protein caused the degradation of p53 through the ubiquitin pathway, through which the function of negative cell cycle control from p53 was abolished, while interaction between E7 and pRb disrupts the ability of pRb to bind E2F cellular transcription factors, and inhibits pRb-mediated repression of E2F-responsive genes, the transcription of downstream genes cause the cell passing the G1/S checkpoint and entering cell cycle. The oncoprotein of HPV can also serve as tumor associate antigen, which stimulate specific immune response in hosts. In order for investigating the mechanism of cell transformation caused by E7, screening the pattern of immune response induced by type specific E7 in the patients and its immunobiological behavior,In this experiment, the HPV58 E7 was expressed by using genetic engineering technique.Methods: Using HPV58 genome as the template,HPV58E7 fragment was amplified By polymerase chain reaction, the pGEX-4T3/HPV58E7 recombinant plasmid was constructed by inserting the HPV58E7 fragment into pGEX-4T3,the GST-tagged expression plasmid and BL21 strain as the host cell. After induction by IPTG, soluble GST-E7 protein was expressed and purified with glutathione sepharose 4B affinity chromatograph.Results: The expressed GST-HPV58E7 protein with 40KD was revealed by SDS-PAGE and purified HPV58E7 without GST-tag was accomplished by digesting with thrombin at the thrombin cleavage site.Conclusion: HPV58E7 protein was efficiently produced with GST-tagged expression system. The products may be used in screening the pattern of immune response evoked by HPV58E7 and the target proteins interacted with E7...