B7-H1慢病毒的制备及其在B16F10细胞中的表达鉴定  

Construction of recombinant lentivirol expression vector carrying mouse B7-H1 gene and its expression and identification in B16F10 cells

作　　者：张立群[1] 段文元[1] 许雪青[1] 黄钢[1] 陈雪丹[1] 白云[1] 

机构地区：[1]第三军医大学基础医学部医学遗传学教研室,重庆400038

出　　处：《第三军医大学学报》2007年第1期24-27,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30271246)~~

摘　　要：目的构建小鼠B7-H1分子慢病毒表达载体并包装成感染性病毒颗粒,获得稳定表达B7-H1的B16F10细胞。方法以小鼠B7-H1的测序质粒为模板,PCR扩增B7-H1全长,装入pLenti6/V5-D-TOPO慢病毒表达载体,经过测序证实其序列与标准序列完全一致。将B7-H1表达载体用L ipofectam ineTM2000转染293FT细胞,包装成感染性病毒颗粒,感染B16F10细胞后,加入含B lastic id in(终浓度为4.0μg/m l)的RPM I1640培养基,筛选出稳定表达B7-H1蛋白的B16F10细胞株。结果PCR扩增得到编码小鼠B7-H1的877 bp基因片段,与预期大小一致。B7-H1重组慢病毒感染B16F10细胞后,用B7-H1单抗进行免疫组化检测,荧光显微镜观察显示B7-H1分子表达于B16F10细胞。FACS测定B16F10细胞荧光阳性率为36%。结论成功构建了B7-H1慢病毒表达载体,包装出感染性病毒颗粒,重组慢病毒感染B16F10细胞后表达出有活性的B7-H1膜表面蛋白,为进一步研究B7-H1分子在器官移植、自身免疫性疾病以及肿瘤免疫逃逸机制中的作用奠定了基础。Objective To construct lentivirol expression vector of mouse B7-H1, and identify its expression in B16F10 cells. Methods Mouse B7-H1 encoding sequence was amplified, purified, ligated with pLen- ti6/V5 lentivirol vector and verified by sequencing. The verified recombinant was transfected into 293FT cells by LipofectamineTM 2000 reagent. The supernatant of the cultured 293FT cells was collected and used to infect B16F10 cells. B16F10 cell line stably expressing B7-H1 protein was selected in the presence of Blasticidin （4.0μg/ml）. Results The mouse B7-H1 gene was successfully amplified by PCR. The recombinant lentivirol vector carrying B7-H1 gene for expression was constructed and its length was about 877 bp, very close to the expected value. By transfecting package cell line 293FT, the recombinant of pLenti6/VS-D-TOPO lentivirol vector carrying B7-H1 infected B16F10 ceils in selective medium. B16F10 transgenic cells could stably express mouse B7-H1 protein on cell membrane. Conclusion Successful construction of mouse B7-H1 lentivirol expression vector and expression of its functional fusion protein based further investigation of the role of B7-H1 in immune tolerance, autoimmune disease and tumor immune escape.

关 键 词：B7-H1 慢病毒 基因治疗 

分 类 号：R373[医药卫生—病原生物学] R392-33[医药卫生—基础医学]