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目的构建人乳头瘤病毒11型(HPV11)E7蛋白基因腺病毒载体,并在真核细胞表达。方法用PCR法,扩增出HPV11E7基因,定向克隆至pENTR-TOPO载体形成重组质粒TOPO-E7。TOPO-E7与腺病毒载体pAD/CMV/V5-DESTTM重组反应,E7基因被重组到腺病毒载体上。该载体经PacI酶切,脂质体法转染人胚肾293A细胞,获得重组腺病毒载体pAD-E7。pAD-E7转染人HaCaT细胞,激光共聚焦显微镜分析E7蛋白表达情况。结果经酶切及序列分析鉴定,重组质粒TOPO-E7成功构建。重组子pAD-E7转染293A细胞后,获得滴度为1.4×107pfu/mL的重组腺病毒载体。该载体转染HaCaT细胞,48h后激光共聚焦显微镜下可见细胞内E7蛋白表达。结论重组腺病毒载体能有效介导HPV11E7基因在真核细胞的表达。
Objective To construct human papillomavirus type 11 (HPV11) E7 protein gene adenovirus vector and express it in eukaryotic cells. Methods HPV11E7 gene was amplified by PCR and cloned into pENTR-TOPO vector to form recombinant plasmid TOPO-E7. TOPO-E7 was recombined with the adenovirus vector pAD / CMV / V5-DESTTM and the E7 gene was recombined into the adenovirus vector. The vector was digested with PacI and transfected into human embryonic kidney 293A cells by liposome to obtain the recombinant adenoviral vector pAD-E7. Human HaCaT cells were transfected with pAD-E7, and the expression of E7 protein was analyzed by laser scanning confocal microscopy. Results After digestion and sequence analysis, the recombinant plasmid TOPO-E7 was successfully constructed. After transfection of recombinant pAD-E7 into 293A cells, a recombinant adenovirus vector with a titer of 1.4 × 107 pfu / mL was obtained. The vector was transfected into HaCaT cells, and the expression of intracellular E7 protein was observed under confocal laser scanning microscope 48h later. Conclusion The recombinant adenovirus vector can effectively induce the expression of HPV11E7 gene in eukaryotic cells.