Cloning And Expression Of HPV16 E5 Gene

Objective Humanpapilloma virus(HPV) is a rigorous philo-epithelium virus. Human being is the only host. Now many studies indicate that HPV is relate to benign or malignant tumor of cervix. Other organs, are reportedly easy to be infected with HPV,including oral mucosa, esophagus,throat,trachea,conjunctiva and anal region. High- risk HPV often has close relation with squamous cell malignant tumor. Most studies on HPV deal mainly with E6 and E7, but seldom with E5. A recent study showed that E5 was often included in high-risk HPV, but seldom found in low-risk HPV. This finding suggests that E5 play an important role in carcinogenesis of HPV. In this paper, HPV E5 is to be cloned and prokaryotic expressed and then the eukaryotic expression vector is to be constructed to transform engineering bacteria. This work will provide the material and theoretic support for further studying the function of HPV16 E5.Methodâ‘ Polymerase chain reaction (PCR) and cloning: primers designed by comparing HPV complete sequences were used in PCR to amplify fragment which contains HPV E5 gene from HPV genome. Amplified fragment was ligated into pMD18-T vector to construct cloning plasmid, then cloning and sequencing were performed. Primers designed again with BamhI, Hindâ…¢endonuclease sites were used in PCR to amplify HPV E5 gene. HPV E5 was ligated into pGEM-T vector to construct cloning plasmid named as pGEM-T-E5, which was identified by Bamh I and Hindâ…¢digestion as well as sequencing.â‘¡Construction of prokaryotic expression vector: digested by restriction enzymes Bamh I and Hindâ…¢, HPV E5 fragment and expression vector pET32a(+) were ligated to construct recombinant prokaryotic expression vector named as pET32a-E5 then was identified by BamhI and Hindâ…¢digestion as well as sequencing.â‘¢Induction of E5-fusion protein: a single colony of E.coli cells transfected with pET32a-E5 was picked and inoculated LB medium and cultured on rocking bed over night,then IPTG was added to induce fusion protein expression. Subsequently,the culture fluid was collected, the cell were lysed using a sonicator, and centrifuged to separate supernatant and pellet. Finally,the products were analyesd by SDS-PAGE and assessed by Western-blotting.â‘£Construction of eukaryotic expression vector: primers designed with Hindâ…¢, BamhI endonuclease sites were used in PCR to amplify HPV E5 gene. HPV E5 fragment and expression vector pLEGFP-N1 were ligated to construct recombinant eukaryotic expression vector named as pLEGFP-N1-E5 and then was identified by BamhI and Hindâ…¢digestion as well as sequencing.Result The HPV E5 gene was successfully obtained. The cloning plasmid with HPV E5 gene and recombinant prokaryotic expression vector were constructed. Both of them were identified by restriction enzyme digestion and sequencing, which showed that HPV E5 gene had been inserted into the multiple cloning site(MCS) correctly. After induction and lysis of the cells,SDS-PAGE analysis showed a newly appeared, conspicuous stripe (the apparently estimated molecular weight was 30KDa),which represented HPV E5 fusion protein. Western-blotting showed that HPV E5 gene could react with the anti papillomavirus antibody.Conclusionsâ‘ HPV E5 was successfully obtained and identified as HPV 16.â‘¡the cloning plasmid pGEM-T-E5, prokaryotic expression vector pET32a-E5 and eukaryotic expression vector pLEGFP-N1-E5 were successfully constructed.â‘¢Fusion protein of E5 was expressed in E.coli BL21 and could react with anti papillomavirus antibody, which might be used to detect the specific antibody. In this study, the systematic methods acquiring HPV E5 fusion protein was established which may help to further study the molecular biological characteristic of the HPV E5.