摘要
目的研究HPV16E7蛋白的生物学特性及其细胞转化机制,对HPV16E7蛋白原核表达质粒进行构建、表达和鉴定。方法以临床确诊的HPV16感染患者子宫颈细胞DNA为模板,采用PCR方法扩增HPV16E7基因,经BamH和Hind双酶切后插入相同酶切pET32a(+)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Westernblot检测目标蛋白表达情况。结果成功构建了原核表达质粒pET32/E7,HPV16E7-TRX融合蛋白在BL21(DE3)菌株中高效表达,在1mmol/LIPTG、30℃诱导条件下融合蛋白表达量占菌体总蛋白的30%左右。结论pET32/E7原核表达质粒的构建、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 construct the prokaryotic expression plasmid pET32/E7 and express the human papillomavirus type 16 E7 protein in E. coll. Methods HPV16 E7 gene was amplified by PCR. The amplified E7 fragment was inserted into the plasmid pET32a （＋） that was digested with BamH Ⅰ and Hind Ⅱ The recombinant plasmid pET32/E7 was transformed into E. coli JM109 which was selected with ampicillin. The positive clones containing recombinant plasmid pET32/E7 were verified by BamH Ⅰ and Xho Ⅰ digestion, and then sequenced. HPV16 ET-TRX recombinant protein expression in the E. coli BL21 （ED3） was identified by SDSPAGE and Western blot. Results The prokaryotic recombinant plasmid pET32/E7 was successfully constructed. The BL21 （DE3） transformed recombinant plasmid pET32/E7 had expressed HPV16 ET-TRX recombinant protein effectively. Under the conditions of 1 mmol/L IPTG and 30 ℃, the amount of HPV16 ET-TRX recombinant protein was about 30% of bacterial total proteins. Conclusion The construction of the prokaryotic recombinant plasmid pET32/E7 and the successful expression of the recombinant protein HPV16 E7-TRX would strongly promote the research of the biological properties and the transformational mechanism of the HPV16 E7 protein on the specific cells.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2006年第2期187-190,共4页
Journal of Sichuan University(Medical Sciences)
关键词
HPV16
原核表达
SDS—PAGE
免疫印迹
Human papillomavirus type 16（HPV16） Prokaryotic Expression SDS-PAGE Immunoblot
