Human papillomavirus E6E7 Construction and identification of recombinant adenovirus.docVIP

 PAGE \* MERGEFORMAT 13 Human papillomavirus E6E7 Construction and identification of recombinant adenovirus Author: Pan Wei-wei, Cheng Hein, Lai flag-prone flat, MA Yong-ping, SONG Fang-zhou [Keywords:] human papilloma virus, human; Adenoviridae Construction and identification of recombinant adenoviruses carrying HPV16 E6E7 [Abstract] AIM: To construct the replicationdeficient recombinant adenoviruses carrying human papillomavirus 16 (HPV16) E6E7 and identify the vector with Western Blot. METHODS: The HPV16 E6E7 gene fragment was obtained from the plasmid pET32a () E6E7 by amplification and digestion, and the shuttle plasmid pAdTrackCMVE6E7 in which the E6E7 was inserted into the downstream of CMV promoter was established by ligation.Then the linearized shuttle plasmid was cotransformed into BJ5183 bacteria with backbone vector AdEasy1 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 replicationdeficient AdE6E7 virus was purified by CsCl density gradient centrifugation. The expression of E6E7 was verified by Western Blot in 293 cell after infected with AdE6E7. RESULTS: The recombinant plasmid pAdE6E7 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 pAdE6E7 in 293 cells and 6.9 * 1010 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. [Keywords] papillomavirus, human; adenoviridae [Abstract] Objective: AdEasy system builds HPV 16 (HPV16) E6 E7 gene recombinant adenovirus