南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (01): 31-35.

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HPV16 E5蛋白原核表达、鉴定及真核表达稳定株的筛选

施桥发;魏晓丽;李虹;王保宁;张卫东;蒋忠华;曹康;李明远;   

  1. 四川大华西基础医学与法医学院微生物学教研室； 成都医学院病原生物学教研室； 四川大学华西基础医学与法医学院微生物学教研室 四川 成都 610041； 四川 成都 610041 新疆医科大学基础医学院免疫学教研室； 新疆 乌鲁木齐 830054； 四川 成都 610041； 四川 成都 610083；
  • 出版日期:2006-01-20 发布日期:2006-01-20
  • 基金资助:
    国家自然科学基金(39970830)

Prokaryotic expression and identification of human papillomavirus type 16 E5 protein

SHI Qiao-fa, WEI Xiao-li, LI Hong, WANG Bao-ning, ZHANG Wei-dong, JIANG Zhong-hua, CAO Kang, LI Ming-yuan Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China; Department of Immunology, Xinjiang Medical University, Urumchi 830054, China; Department of Microbiology, Chengdu Medical College, Chengdu 610083, China   

  1. 四川大华西基础医学与法医学院微生物学教研室； 成都医学院病原生物学教研室； 四川大学华西基础医学与法医学院微生物学教研室 四川 成都 610041； 四川 成都 610041 新疆医科大学基础医学院免疫学教研室； 新疆 乌鲁木齐 830054； 四川 成都 610041； 四川 成都 610083；
  • Online:2006-01-20 Published:2006-01-20

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摘要： 目的研究HPV16 E5蛋白的生物学特性及其细胞转化机制,对HPV16 E5蛋白原核和真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HPV16 E5基因,经 BamH Ⅰ和HindⅢ双酶切后插入相同酶切的pET32a(+)载体,转染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Westem blotting检测目标蛋白表达情况。将BamH Ⅰ和Xho Ⅰ双酶切 pET32(+)／E5质粒后取得的E5全基因片段转入pcDNA3．1(+)空质粒,构建pcDNA3．1(+)／E5真核表达质粒并对NIH3T3 细胞进行转染,最后用G418进行稳定表达株的筛选,并采用RT-PCR鉴定细胞内HPV16 E5基因表达情况。结果成功构建了原核表达质粒pET32／E5,在1 mmol／L IPTG、28℃诱导条件下BL21(DE3)菌体中HPV16E5-TRX融合蛋白占菌体总蛋白的10％左右。真核表达质粒pcDNA3．1(+)／E5在成功转染NIH3T3细胞后于250μg/ml G418浓度时筛选21 d得到了E5基因稳定表达株,RT-PCR产物测序得到了HPV16 E5基因全序列。结论成功构建了pET32／E5原核和 pcDNA3．1(+)／E5真核表达质粒,HPV16E5蛋白在大肠杆菌和NIH3T3细胞中的稳定表达,这些结果为进一步深入研究HPV16 E5蛋白的生物学特性及其致细胞转化的作用奠定了坚实基础。

Abstract: Objective To construct the prokaryotic and eukaryotic expression vectors pET32/E5 and pcDNA3.1/E5 for transformation into E. coli BL21 and NIH3T3 cells respectively to observe the expression of human papillomavirus type 16 E5 protein (HPV16 E5). Methods HPV16 E5 gene was amplified by PCR from clinical isolates of HPV 16 and inserted into the plas-mid pET32a(+) followed by digestion with BomH Ⅰ and Hind Ⅲ. The recombinant plasmidpET32/E5 was transformed into E. coli JM109 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32/E5 were verified by restriction endonucleases BomH I and Xho and sequence analysis. The expression of HPV16 E5-TRX fusion protein in E.coli BL21(ED3) was identified by SDS-PAGE and Western blotting. The digestion product of BamH I and Xho I was purified and inserted into the eukaryotic expression vector pcDNA3.1(+) to construct pcDNA3.1/E5, which was identified by sequencing and transfected into NIH3T3 cells. The NIH3T3 cells with stable expression of HPV16 E5 were selected by G418 and confirmed by RT-PCR. Results The pET32/E5 and pcDNA3.1/E5 vectors were constructed successfully. E.coli BL21(DE3) transformed by the recombinant plasmid pET32/E5 expressed HPV16 E5-TRX fusion protein efficiently. In the presence of 1 mmol/L IPTG at 28 ℃, HPV16 E5-TRX recombinant protein accounted for about 10% of the total bacterial proteins. NIH3T3 cells stably expressing HPV16 E5 were harvested by selection with 250 g/ml of G418. HPV16 E5 gene from pcDNA3.1/E5-transfect-ed NIH3T3 cells was amplified by RT-PCR, and sequence analysis demonstrated the acquisition of the full-length gene fragment. Conclusions The prokaryotic and eukaryotic vectors for the HPV16 E5 gene have been successfully constructed. The acquisition of E.coli and NIH3T3 cells with stable expression of HPV 16 E5 protein may facilitate subsequent research of the biological properties and the transformation mechanism of HPV16 E5 protein on specific cells.

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