人乳头瘤病毒6bL1双顺反子载体的构建及其在哺乳动物细胞内的表达

Construction of a bicistronic expression vector containing human papillomavirus （HPV） type 6b L1 gene and its expression in mammalian cells

目的构建尖锐湿疣患者人乳头瘤病毒6b型晚期基因（HPV6bLI）的双顺反子表达载体，并使其在哺乳动物细胞内表达，以期建立含有HPV6bLI的细胞模型。方法表达质粒pEGFP—HPV6bL1经双酶切纯化后与经过相同双酶切的真核表达质粒pIRES2-EGFP连接，酶切鉴定，挑选阳性克隆进行测序。重组质粒pIRES2-HPV6bL1-EGFP转染进小鼠成纤维细胞（NIH3T3），荧光显微镜下观察EGFP蛋白的表达，RT—PCR检测HPV6b L1 mRNA的生成。结果成功构建含HPV6bL1的重组质粒pIRES2-HPV6bL1-EGFP。重组体成功转染进NIH3T3细胞，并用G418筛选。同时荧光倒置显微镜下可观察到细胞内有绿色荧光蛋白的表达。进一步进行RT—PCR，检测到HPV6bL1mRNA的生成。结论成功构建携带HPV6bL1的重组体pIRES2-HPV6bL1-EGFP并转染入NIH3T3细胞。经荧光倒置显微镜观察及RT—PCR方法检测证明HPV6bLI在NIH3T3细胞内成功表达。

Objective To construct a bicistronic expression vector containing HPV type 6b L1 gene, to express the recombinant vector in mammalian cells, and to establish a cell strain stably expressing HPV6b L1 gene. Methods After endonuclease digestion and purification, the gene fragment of HPV6b L1 was cloned into the eukaryotic expression vector pIRES2-enhanced green fluorescent protein （EGFP）. The identification of the recombinant was realized via endonuclease digestion and sequence analysis. Then, the recombinant plasmid plRES2-HPV6bL1-EGFP was transfected into NIH3T3 （a mouse embryonic fibroblast cell line） cells. Subsequently, the expression of EGFP was observed by fluorescent inverted microscopy, and HPV6b L1 mRNA expres- sion by reverse transcription （RT）-PCR. Results The recombinant plasmid pIRES2-HPV6bL1-EGFP was successfully constructed, transfected into NIH3T3 cells, and selected by G418. The expression of EGFP was seen under an inverted fluorescence mieroscoy. RT-PCR proved the expression of HPV6b L1 mRNA in transleered cells. Conclusions The recombinant plasmid pIRES2-HPV6bL1-EGFP was successfully constructed and transfected into NIH3T3 cells. Inverted fluorescent microscopy and RT-PCR confirmed the successful expression of HPV6b L1 in NIH3T3 cells.