Construction and identification of recombinant adenoviruses carrying HPVª²16 E6E7

    PAN Weiª²Wei, CHENG Haiª²En, LAI Guoª²Qi, YI Faª²Ping, MA Yongª²Ping, SONG Fangª²Zhou

    Department of Biochemistry and Molecular Biology, Laboratory Animal Centre, Key Laboratory of Ministry of Education for Clinical Diagnostics, 4Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology, Chongqing University of Medical Sciences, Chongqing 400016, China

    ¡¾Abstract¡¿ AIM: To construct the replicationª²deficient recombinant adenoviruses carrying human papillomavirus 16(HPVª²16)E6E7 and identify the vector with Western Blot. METHODS: The HPVª²16 E6E7 gene fragment was obtained from the plasmid pETª²32a(+)ª²E6E7 by amplification and digestion, and the shuttle plasmid pAdTrackª²CMVª²E6E7 in which the E6E7 was inserted into the downstream of CMV promoter was established by ligation.Then the linearized shuttle plasmid was coª²transformed into BJ5183 bacteria with backbone vector AdEasyª²1 to obtain the recombinant adenoviral plasmid pAd E6E7 by homologous recombination. The recombined adenovirus DNA was transfected into 293 cells for packing and the amplification product of replicationª²deficient Adª²E6E7 virus was purified by CsCl density gradient centrifugation. The expression of E6E7 was verified by Western Blot in 293 cell after infected with Adª²E6E7. RESULTS: The recombinant plasmid pAdª²E6E7 was established by homologous recombination and confirmed by restriction endonuclease digestion and sequencing. GFP expression could be observed 3 d after packing of the linearized pAdª²E6E7 in 293 cells and 6.9¡Á1010 pfu/L titer of Ad E6E7 was obtained by CsCl gradient purification. The 293 cell was infected with this titer of Ad E6E7 viruses, and then total protein in 293 cells was extracted. Western Blot showed that E6E7 protein was expressed obviously. CONCLUSION: Ad E6E7 expressing both GFP and E6E7 simultaneously can be simply and rapidly generated by using the AdEasy system. ......