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目的构建含人乳头状瘤病毒16型E7(HPV16E7)基因的重组腺相关病毒(rAAV)载体,并验证HPV16E7mRNA和蛋白在真核细胞中的表达。方法将含腺相关病毒(AAV)末端反向重复序列及HPV16E7基因的重组质粒pAAVMCSE7、含rep/cap基因的质粒pAAVRC及辅助质粒pAAVhelper共同转染胚胎肾细胞系HEK293细胞,回收、纯化病毒颗粒并鉴定,斑点杂交法测定病毒滴度,RTPCR技术、蛋白印迹法验证HPV16E7mRNA和蛋白在真核细胞中的表达。结果电镜鉴定结果显示真核细胞中有病毒颗粒存在,斑点杂交法测定病毒滴度为1×1011/ml。含HPV16E7基因的rAAV载体转染真核细胞后,在细胞内可检测到HPV16E7mRNA和蛋白的表达。结论本实验成功构建了含HPV16E7基因的rAAV载体,经验证HPV16E7mRNA和蛋白在真核细胞内有表达。
Objective To construct recombinant adeno-associated virus (rAAV) vector containing human papillomavirus type 16 E7 (HPV16E7) gene and verify the expression of HPV16E7 mRNA and protein in eukaryotic cells. Methods Recombinant plasmid pAAVMCSE7 containing the reverse transcription sequence of adeno-associated virus (AAV) and HPV16E7 gene, plasmid pAAVRC containing rep / cap gene and pAAVhelper helper plasmid were co-transfected into embryonic kidney cell line HEK293, and the virus particles were recovered and purified Identification, dot blot hybridization to determine the virus titer, RTPCR technology, western blotting to verify HPV16E7mRNA and protein expression in eukaryotic cells. Results Electron microscopy showed that there were virus particles in eukaryotic cells. The virus titer was 1 × 1011 / ml as determined by dot blot hybridization. After transfection of eukaryotic cells with rAAV vector containing HPV16E7 gene, HPV16E7 mRNA and protein expression can be detected in cells. Conclusion The rAAV vector containing HPV16E7 gene was successfully constructed in this experiment. The expression of HPV16E7 mRNA and protein in eukaryotic cells was verified.