摘要
目的:在果蝇S2细胞中表达人乳头瘤病毒16型(HPV-16)E1。方法:PCR扩增HPV-16 E1全长,将PCR产物连接至pMD18-T并测序鉴定,继而将HPV-16 E1构建至果蝇表达载体pMT/Bip/V5-HisA中。大量提取pMT/Bip/V5/His-E1重组表达载体并与筛选质粒pCoBlast共转染果蝇S2细胞,经杀稻瘟菌素(Blasticidin S)筛选获得具有抗性的稳定转染S2细胞。提取稳转S2细胞基因组DNA,PCR鉴定S2细胞中整合的E1。以终浓度为5μmol/L CdCl2诱导表达,收集上清进行SDS-PAGE及Western blot鉴定。结果:双酶切及测序结果显示HPV-16 E1基因已克隆人重组质粒pMT/Bip/V5-E1,PCR和Western blot结果表明HPV-16 E1基因已整合至果蝇S2细胞基因组并稳定表达。结论:获得HPV-16 E1转染的果蝇S2细胞株,该细胞株可持续稳定表达E1蛋白。
Objective：To express human papillomavirus type 16 （HPV -16） E1 in Drosophila S2 cells. Method：HPV -16 E1 was ampli- fied by PCR and cloned into pMD18 - T vector,then the HPV - 16 E1 was cloned into Drosophila Schneider expression vector pMT/Bip/ V5 - HisA. The recombinant plasmid pMT/Bip/V5/His - E1 and pCoBlast were co - transfected into Drosophila S2 cells, the S2 cells were screened by blasticidin （Blasticidin S）. The genomie DNA was extracted from the transfected S2 cells, then identified by PCR. The transfeeted cells were induced with 5μmol /L CdC12 , then the supernatant was collected and detected by SDS - PAGE and Western blot. Result：Restriction endonuclease analysis and DNA sequencing showed HPV - 16 E1 was cloned into the plasmid pMT/Bip/V5 - El. The results of PCR and Western blot indicate that HPV - 16 E1 was integrated into S2 cells and was expressed stably in the transfected cells. Conclusion：The S2 cell line transfeeted with HPV- 16 El was achieved, and E1 express stably and sustainably in the cell line.
出处
《生物技术》
CAS
CSCD
北大核心
2014年第4期17-20,共4页
Biotechnology
基金
国家自然科学基金项目("新疆地区人乳头状瘤病毒16型上游调控区突变体启动子活性和复制功能的研究"
No.31060025)
新疆生物资源基因工程重点项目(No.XJDX0201-2013-01)资助
