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目的构建人乳头瘤病毒(HPV)E6、E7基因慢病毒真核表达载体。方法以Si Ha宫颈癌细胞总RNA为模板,利用RT-PCR技术扩增出E6、E7目的片段;选用病毒载体p Lenti6/V5 Directional TOPO Cloning Kit系统构建E6、E7基因慢病毒真核表达载体;分别使用构建好的E6、E7载体转染病毒包装细胞,获得含病毒的上清液,并测定上清液中慢病毒的滴度。结果质粒测序所得序列与人乳头瘤病毒E6、E7基因序列完全吻合,载体构建成功;慢病毒滴度测定结果显示E6样品的慢病毒滴度为1.5×108TU/m L,E7样品的慢病毒滴度为2×108TU/m L,该滴度能够满足后续研究所需。结论 HPV-16 E6、E7基因慢病毒真核表达载体构建成功,可以为进一步研究人乳头瘤病毒E6、E7基因致病机理奠定物质基础。
Objective To construct human papillomavirus (HPV) E6, E7 lentivirus eukaryotic expression vector. Methods E6 and E7 fragments were amplified by RT-PCR from total RNA of Si Ha cervical cancer cells. The E6 and E7 lentiviral eukaryotic expression vectors were constructed by using the p Lenti6 / V5 Directional TOPO Cloning Kit. The constructed E6 and E7 vectors were respectively used to transfect the virus-packed cells to obtain the virus-containing supernatant and the titer of the lentivirus in the supernatant was measured. Results The sequence of plasmid sequencing was completely consistent with the sequence of human papillomavirus E6 and E7, and the vector was constructed successfully. The titer of lentivirus showed that the lentiviral titer of E6 was 1.5 × 108TU / m L, The degree of 2 × 108TU / m L, the titer to meet the needs of the follow-up study. Conclusion The eukaryotic expression vector of HPV-16 E6 and E7 lentivirus was successfully constructed and could lay a material foundation for further study on the pathogenesis of human papillomavirus E6 and E7 genes.