稳定表达人乳头瘤病毒(HPV)58型E6E7融合基因的B16细胞系的建立  

Establishment and identification of B16 cell line stably expressing human papillomavirus(HPV) type 58 E6E7 fusion gene

作　　者：王鹤[1] 于继云[2] 李力[1] 

机构地区：[1]广西医科大学附属肿瘤医院,南宁530021 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《中国免疫学杂志》2011年第12期1088-1092,共5页Chinese Journal of Immunology

基　　金：2009年广西卫生厅重点课题资助项目(重200970)

摘　　要：目的:构建pIRES-neo-HPV58E6E7真核表达载体,稳定转染入小鼠黑色素瘤B16细胞,建立稳定表达HPV58E6E7基因的B16细胞系。方法:采用PCR方法扩增出HPV58E6E7融合基因的全长序列,利用DNA重组技术将其定向插入到真核表达载体pIRES-neo中,并加入酶切位点和6×his标签,构建重组真核表达质粒pIRES-neo-HPV58E6E7。利用阳离子脂质体介导法将其转染入小鼠黑色素瘤B16细胞,经G418加压筛选出稳定转染的阳性克隆。利用Western blot、流式细胞术、免疫荧光等检测方法验证HPV58E6E7融合基因在稳定转染的B16细胞株中的表达。结果:经PCR、限制性内切酶鉴定及测序分析,pIRES-neo-HPV58E6E7重组质粒构建正确,转染B16细胞株后,经过Western blot、流式细胞术和免疫荧光等检测显示B16细胞株能够稳定、高效表达HPV58E6E7融合基因,表明B16-HPV58E6E7稳定转染细胞系构建成功。结论:成功构建了pIRES-neo-HPV58E6E7真核表达载体,建立了可以高效、稳定表达HPV58E6E7融合基因的B16细胞系。该稳定转染细胞系的建立为进一步研究HPV58治疗性基因疫苗的功能提供了良好的靶细胞,为其在肿瘤免疫治疗中的应用奠定了研究基础。Objective：To construct the eukaryotic expression vector of HPV58 E6E7 fusion gene and stably transfect B16 cell line with it.Methods：The full length of HPV58E6E7 fusion gene fragment was amplified by PCR and inserted into eukaryotic expression vector pIRES- neo, added the restriction enzyme position and 6 × his tag. After identification of restriction digestion and PCR, The recombinant plasmid pIRES-neo-HPV58E6E7 was obtained. Then transfected it into B16 cells by lipofectamine 2000. After screening culture by G418, a stably transfected cell line was established, the transcription and expression of the HPV58E6E7 fusion gene were identified by Western blot, FACS and im- munofluorescence assay. Results： The eukaryotic expression vector pIRES-neo-HPV58E6E7 was successfully constructed. The expression of HPV58E6E7 fusion gene was fotmd positive by Western blot,FACS and immunofluorescence,＇indicating that the B16 cell line stably and highly expressing I-IPV58E6E7 fusion gene was successfully established. Conclusion： The established cell line B16-HPV58E6E7 can highly and stably express HPV58 E6E7 fusion gene, the expression of the target gene provide a solid experimental foundation for further studies on the function of HPV58 gene vaccine,and which will contribute to the research of the target gene in the tumor immunotherapy.

关 键 词：人乳头瘤病毒58型 E6E7基因 稳定转染 小鼠黑色素瘤细胞 

分 类 号：R285.5[医药卫生—中药学]