HPV16E7基因的原核克隆及表达

Prokaryotic cloning and expression of HPV16 E7 gene

目的:探讨从喉癌组织中HPV16E7蛋白的原核克隆及表达。方法:采用PCR方法扩增HPV16E7基因,经BamHⅠ和HindⅢ双酶切后插入相同酶切pET28a(+)载体,转化JM109感受态细胞,并进行阳性克隆筛选。经IPTG对重组质粒进行蛋白诱导表达,SDS-PAGE和Western blot检测目标蛋白表达情况。结果:成功构建了原核表达质粒pET28/E7、HPV16E7融合蛋白在BL21(DE3)菌株中高效表达。结论:HPV16E7融合蛋白的表达为进一步深入研究HPV16E7蛋白的生物学特性及其致细胞转化的作用机制奠定了基础。

Objective：To explore the cloning and expression of HPV16 E7 protein from laryngeal carcinoma. Methods： HPV16 E7 gene was amplified by PCR. The amplified fragment was inserted into the plasmid pET28a （＋） digested with BamHⅠand Hind Ⅲ. The recombinant plasmid pET28/E7 was transformed into E.coli JM109 which was selected with ampicillin. HPV16 E7 recombinant protein expression in the E.coli BL21（DE3） was identified by SDS-PAGE and Western blot. Results： The prokaryotic recombinant plasmid pET28/E7 was successfully constructed. The BL21（DE3） transformed recombinant plasmid pET28/E7 had expressed HPV16 E7 recombinant protein effectively. Conclusion：The construction of the prokaryotic recombinant plasmid pET28/E7 and the successful expression of the recombinant protein HPV16 E7 pave way for the profound research of the biological properties and the transformational mechanism of the HPV16 E7 protein on the specific cells.