人乳头瘤病毒E6E7基因重组腺病毒的构建及鉴定

Construction and identification of recombinant adenoviruses carrying HPV-16 E6E7

目的:利用AdEasy系统构建人乳头瘤-16(HPV-16)E6E7基因重组腺病毒,并通过WesternBlot方法检测E6E7蛋白的表达.方法:将质粒pET-32a(+)-E6E7扩增、酶切获得E6E7片段插入腺病毒穿梭载体质粒pAdTrack-CMV的巨细胞病毒(CMV)启动子下游,构建重组穿梭载体pAdTrack-CMV-E6E7,线性化后与骨架载体AdEasy-1在细菌BJ5183内同源重组得到腺病毒质粒pAd-E6E7,经人胚肾293细胞包装后得到复制缺陷型重组腺病毒Ad-E6E7;用包装后的病毒上清再次感染293细胞,提取细胞中的蛋白,通过WesternBlot方法检测E6E7蛋白的表达.结果:连接、重组后通过酶切和测序法筛选出pAd-E6E7;经人胚肾293细胞包装,3d后观察到绿色荧光蛋白(GFP)明显表达,氯化铯梯度离心纯化最终获得6.9×1010pfu/L滴度的重组病毒;用该滴度的Ad-E6E7重新感染人胚肾293细胞3d后,提取细胞蛋白,WesternBlot检测,E6E7有明显表达.结论:利用新型腺病毒载体AdEasy系统可在短期内制备同时表达GFP和E6E7的重组腺病毒AdE6E7.

AIM： To construct the replication-deficient recombinant adenoviruses carrying human papillomavirus 16 （HPV-16） E6E7 and identify the vector with Western Blot. METHODS： The HPV-16 E6E7 gene fragment was obtained from the plasmid pET-32a （ ＋ ）-E6E7 by amplification and digestion, and the shuttle plasmid pAdTrack-CMV-E6E7 in which the E6E7 was inserted into the downstream of CMV promoter was established by ligation. Then the linearized shuttle plasmid was co-transformed into BJ5183 bacteria with backbone vector AdEasy-1 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 Ad-E6E7 virus was purified by CsCI density gradient centrifugation. The expression of E6E7 was verified by Western Blot in 293 cell after infected with Ad-E6E7. RESULTS： The recombinant plasmid pAd-E6E7 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 pAd-E6E7 in 293 cells and 6.9 × 10^10 pfu/L titer of Ad E6E7 was obtained by CsCI 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.