构建及鉴定携带角蛋白启动子和人乳头瘤病毒16E6/E7基因的重组腺病毒

目的利用AdEasy系统构建携带人角蛋白启动子的人乳头瘤病毒-16(HPV-16)E6/E7基因重组腺病毒,并通过RT-PCR方法检测E6/E7基因的表达。方法应用PCR方法从含有HPV-16全基因序列的质粒上扩增E6/E7基因,构建pCDNA3.1(-)-K14-E6/E7-polA载体,扩增、酶切获得K14-E6/E7-polA片段插入腺病毒穿梭载体质粒pAdTrack上,构建重组穿梭载体pAdTrack-K14-E6/E7-polA,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-K14-E6/E7-polA,经人胚肾293细胞包装后得到重组腺病毒pAd-K14-E6/E7-polA。氢化铯(CsCl)梯度离心纯化病毒,提取病毒再感染后的293细胞总RNA,通过RT-PCR方法检测E6/E7基因的表达。结果通过同源重组的方法构建了腺病毒pAd-K14-E6/E7-polA载体,经酶切和测序鉴定该质粒构建成功。293细胞包装3d后观察到绿色荧光蛋白(GFP)表达,CsCl梯度离心纯化最终获得7.2×1010pfu/mL滴度的重组病毒;用该滴度病毒重新感染293细胞3d后,提取细胞总RNA,RT-PCR检测E6/E7有表达。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E6/E7的重组腺病毒pAd-K14-E6/E7-polA。这将为进一步研究HPV-16E6/E7基因功能及利用基因治疗女性宫颈癌奠定了基础。

Construction and identification of recombinant adenoviruses carrying cytokeratin promoters 14 and HPV-16 E6/E7

Objective To construct the recombinant adenoviruses carrying cytokeratin promoters and HPV-16 E6/E7 gene by AdEasy system and to identify the expression of E6/E7 mRNA by RT-PCR. Methods HPV-16 E6/E7 was amplified by polymerase chain reaction(PCR) from vectors that contained all sequence of HPV16, and the pcDNA3.1(-)-K14-E6/E7-polA vector was constructed. The oligo K14-E6/E7-polA was obtained by amplifying and digesting the pcDNA3.1(-)-K14-E6/E7-polA vector. The shuttle plasmid pAdTrack-K14-E6/E7-polA was constructed by inserting the K14-E6/E7-polA into the pAdTrack vector. Then the linearized shuttle plasmids were co-transformed into bacteria containing AdEasy-1 backbone vector for homologous recombination and obtaining the recombinant adenoviral plasmid pAd-K14-E6/E7-polA. The recombined adenovirus DNA was transfected into 293 cells for packing of Ad-K14-E6/E7-polA virus, and the virus was purified by CsCl density gradient centrifugation. The expression of E6/E7 was determined by RT-PCR after infection of Ad-K14-E6/E7-polA in 293 cells. Results The recombinant plasmid pAd-K14-E6/E7-polA was successfully constructed and confirmed by restriction endonuclease digestion and sequencing. GFP expression could be observed on the 3rd day after the package of linearized pAd-K14-E6/E7-polA in the 293 cells. The titer of pAd-K14-E6/E7-polA was 7.2×10~ 10 pfu/mL by CsCl gradient purification. Total RNA in 293 cells was collected 3 days after the 293 cells were re-infected with the pAd-K14-E6/E7-polA virus with the titer being 7.2×10~ 10 pfu/mL. The RT-PCR results indicated that E6/E7 could be detected in the transfected 293 cells. Conclusion The recombinant adenoviruses Ad-K14-E6/E7-polA expressing GFP and E6/E7 simultaneously could be generated in a short time by using the AdEasy system. The construction of recombinant adenoviruses Ad-K14-E6/E7-polA provides a base for further study of the gene function of HPV-16 E6/E7 and gene therapy of female cervical carcinoma.