HPV58 E6基因的克隆及体外表达

CLONING AND IN VITRO EXPRESSION OF HPV58E6 GENE

目的 :克隆并体外表达HPV5 8E6,为E6致癌作用和疫苗的研究奠定基础。方法 :通过PCR方法从HPV5 8全基因克隆中扩增出HPV5 8E6基因片段 ,利用基因重组技术 ,将其克隆至真核表达质粒pcDNA3的T7启动子下游 ,体外转录成mRNA后 ,在源自网织红细胞的翻译缓冲液中表达生物素标记的HPV5 8E6蛋白 ,化学发光法检测 ,X光胶片曝光显影鉴定。结果 :酶切电泳证实质粒构建正确 ,SDS PAGE电泳显示在相当于HPV5 8E6分子量约 18.8kD的位置出现条带 ,X光胶片亦在相应位置出现印迹条带。结论 :成功表达了含有生物素标记的HPV5 8E6蛋白 。

Objective:To clone and in vitro express biotin labeled HPV58E6, and provide a good basis for further research on its function and vaccine.Methods:HPV58E6 gene fragment was amplified from genome of HPV58 by PCR, and cloned into eukaryotic expression vector pcDNA3. The recombinant plasmid was transcripted in vitro and then translated into biotin labeled HPV58E6 protein in rabbit reticulocyte lysate. The protein was detected by chemiluminescence blotting and gave a signal on the X ray film in the end.Results: The recombinant expression plasmid pcDNA3 HPV58E6 was constructed successfully and the E6 protein whose molecular weight is 18.8kD was seen in the SDS PAGE analysis and also gave a signal at the right position on the X ray film.Conclusion: This biotin labeled protein can be used in further research on HPV58E6 function and vaccine development.