论文部分内容阅读
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。
Objective To construct the recombinant adenovirus carrying HPV16 (E6 / E7) carrying human keratin promoter using AdEasy system and detect the expression of E6 / E7 gene by RT-PCR. Methods The E6 / E7 gene was amplified from the plasmid containing the full-length HPV-16 gene by PCR and cloned into pCDNA3.1 (-) - K14-E6 / E7-polA vector. The recombinant shuttle vector pAdTrack-K14-E6 / E7-polA was constructed by inserting the recombinant plasmid pAdA into the adenoviral shuttle plasmid pAdTrack. After homologous recombination with the backbone vector AdEasy-1 in the bacterium BJ5183, the adenovirus plasmid pAd-K14- E6 / E7-polA, recombinant adenovirus pAd-K14-E6 / E7-polA was obtained after packaging by human embryonic kidney 293 cells. The total RNA was extracted from 293 cells after virus re-infection. The expression of E6 / E7 gene was detected by RT-PCR. Results The adenovirus pAd-K14-E6 / E7-polA vector was constructed by homologous recombination. The plasmid was identified by restriction enzyme digestion and sequencing. The expression of green fluorescent protein (GFP) was observed after 293 cells were packaged for 3 days. CsCl gradient centrifugation finally yielded a recombinant virus with a titer of 7.2 × 1010 pfu / mL. After being reinfected with 293T cells for 3 days, total RNA was extracted and RT The expression of E6 / E7 was detected by PCR. Conclusions Recombinant adenovirus pAd-K14-E6 / E7-polA, which can express both GFP and E6 / E7 simultaneously, can be prepared in a short term by using the new adenovirus AdEasy system. This will lay the foundation for further study of HPV-16E6 / E7 gene function and gene therapy of cervical cancer in women.