小鼠Tim-3真核表达载体pTARGET-Tim-3的构建  

Construction of the mouse T cell immunoglobulin mucin 3( Tim-3) eukaryotic expression vector

作　　者：孟静[1] 刘晓立[2] 郭春[3] 马春红[3] 

机构地区：[1]山东大学第二医院检验科,济南250033 [2]山东大学第二医院肾移植科,济南250033 [3]山东大学医学院免疫学研究所,济南250012

出　　处：《山东大学学报（医学版）》2010年第11期37-40,共4页Journal of Shandong University:Health Sciences

基　　金：山东省科技厅攻关计划资助项目(2007GG30002028);山东省卫生厅青年基金资助项目(2007QZ011)

摘　　要：目的构建小鼠Tim-3真核表达载体,并在黑色素瘤细胞系B16中表达小鼠TIM-3。方法以脾细胞RNA为模板,逆转录PCR扩增鼠Tim-3编码区基因,T-A克隆至真核表达载体pTARGET,构建重组质粒pTARGET-Tim-3,采用脂质体转染法转染黑色素瘤细胞系B16细胞,以逆转录PCR和Westernblot验证Tim-3在B16细胞中的表达。结果利用酶切和测序的方法,筛选、鉴定pTARGET-Tim-3真核表达载体,转染黑色素瘤细胞系B16细胞,经逆转录PCR和Westernblot证实TIM-3高效表达。结论成功构建小鼠Tim-3真核表达载体,Tim-3在转染的小鼠B16细胞系中高表达。Objective To construct an eukaryotic expression vector of murine T cell immunoglobulin mucin 3（ Tim-3） , and express Tim-3 in the melanoma cell line B16. Methods The murine Tim-3 coding region was amplified by reverse transcription PCR （ RT-PCR） ,using RNA from spleen cells as a template. The Tim-3 cDNA was cloned into the eukaryotic expression vector pTARGET by T-A cloning. The recombinant vector was transfected into the melanoma cell line B16 by Lipofectamine. Tim-3 expression was analyzed by RT-PCR and Western blot. Results The murine Tim-3 expression vector was identified by enzyme digestion and sequencing. RT-PCR and Western blot results showed specific Tim-3 expression in B16 cells transfected with the recombinant vector. Conclusion The Tim-3 coding region was successfully cloned into the eukaryotic expression vector and highly expressed in the melanoma cell line B16.

关 键 词：基因 克隆 分子 DNA 重组 真核表达 小鼠 

分 类 号：R392.12[医药卫生—免疫学]