Construction and identification of recombinant plasmid pET21 b-HPV16E4

Objective To construct a recombinant plasmid pET21b-HPV16E4 for HPV16 E4 protein expression in E.coli and study function of HPV16 E4 protein.Methods PCR was used to amplify HPV16E4 gene from cervical cancer tissue infected by HPV16.The plasmid,pET21b-HPV16E4,was then constructed by inserting the amplified HPV16 E4 gene into pET21b,a prokaryotic expression vector.PCR was used to screen the positive recombinant clones.Restriction analysis and sequencing were used to confirm the construction of pET21b-HPV16E4.Results HPV16E4 gene(0.3kb) was amplified and recombinated into pET21b vector.The new plasmid,pET21b-HPV16E4,was identified by PCR and restricting enzyme digestions.Two fragments,0.3kb and 5.4kb,could be obtained by BamHⅠ and HindⅢ digestions.Sequencing further verified the recombinant.Conclusion The plasmid pET21b-HPV16E4 is constructed successfully.