Shi Qiao-Fa, Wang Bao-Ning, Li Hong, Wei Xiao-Li, Zhang Wei-Dong, Cao Kang, Jiang Zhong-Hua, Li Ming-yuan
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2006 Mar;37(2):187-90.
To construct the prokaryotic expression plasmid pET32/E7 and express the human papillomavirus type 16 E7 protein in E. coli.
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 III. 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 E7-TRX recombinant protein expression in the E. coli BL21(ED3) was identified by SDS-PAGE and Western blot.
The prokaryotic recombinant plasmid pET32/E7 was successfully constructed. The BL21(DE3) transformed recombinant plasmid pET32/E7 had expressed HPV16 E7-TRX recombinant protein effectively. Under the conditions of 1 mmol/L IPTG and 30 degrees C, the amount of HPV16 E7-TRX recombinant protein was about 30% of bacterial total proteins.
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.
构建原核表达质粒pET32/E7，并在大肠杆菌中表达人乳头瘤病毒16型E7蛋白。
采用PCR扩增HPV16 E7基因。将扩增的E7片段插入经BamH I和Hind III酶切的质粒pET32a(+)中。将重组质粒pET32/E7转化至用氨苄青霉素筛选的大肠杆菌JM109中。通过BamH I和Xho I酶切鉴定含重组质粒pET32/E7的阳性克隆，然后进行测序。通过SDS-PAGE和Western blot鉴定HPV16 E7-TRX重组蛋白在大肠杆菌BL21(DE3)中的表达情况。
成功构建原核重组质粒pET32/E7。转化重组质粒pET32/E7的BL21(DE3)能有效表达HPV16 E7-TRX重组蛋白。在1 mmol/L IPTG和30℃条件下，HPV16 E7-TRX重组蛋白量约占细菌总蛋白的30%。
原核重组质粒pET32/E7的构建及重组蛋白HPV16 E7-TRX的成功表达将有力推动HPV16 E7蛋白生物学特性及其对特定细胞转化机制的研究。
