人TCA-12-2/TCB-7.1真核双表达载体的构建与表达  

Construction and identification of eukaryotic dicistronic vector expressing TCA-12-2/TCB-7.1

作　　者：张文峰[1] 张巨峰[1] 邵红伟[1] 王俊伟[1] 牟艳芳[1] 黄树林[1] 

机构地区：[1]广东药学院生命科学与生物制药学院生物制药研究所,广东广州510006

出　　处：《中国病理生理杂志》2008年第3期527-531,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30572124);广东省自然科学基金资助项目(No.5002855);广州市科技计划资助项目(No.2004Z3-E4061);广东药学院博士科研启动基金资助项目(No.43555014)

摘　　要：目的:将T细胞抗原受体基因TCA-12-2和TCB-7.1共同构建在真核表达载体pDC315,用于哺乳动物细胞293转染研究。方法:将分离得到的淋巴细胞总RNA反转录合成cDNA,以此为模板进行PCR扩增得到TCA-12-2基因;以实验室保存含TCB-7.1基因的质粒为模板,扩增得到TCB-7.1基因。随后酶切,连入载体pIRES2-AcGFP1,通过亚克隆连入载体pDC315,得到重组质粒pDC315-TCA-12-2-TCB-7.1。重组体质粒经酶切鉴定后,并对插入的TCA-12-2和TCB-7.1基因片段进行测序,将鉴定好的阳性重组质粒用脂质体介导转染293细胞,并通过RT-PCR和流式细胞仪检测TCA-12-2和TCB-7.1基因的表达情况。结果:TCA-12-2和TCB-7.1可以同时构建在载体pDC315上,RT-PCR和流式细胞仪检测发现TCA-12-2和TCB-7.1基因可以成功在293细胞上表达。结论:成功构建了含TCA-12-2和TCB-7.1基因的双表达载体。AIM： To construct the recombinant dicistronic eukaryotic expression vector pDC315 -TCA - 12 - 2 -TCB -7.1, which containing T cell antigen receptor （TCR） genes TCA - 12 -2 and TCB -7.1, and to transfer this recombinant vector into 293 cells to investigate the expression of TCA - 12 -2 and TCB -7.1. METHODS： The TCA - 12 -2 was obtained by RT - PCR from the T cells and the TCB -7.1 was amplified by PCR from plamid pcDNA3.1 -TCB -7.1 that we constructed before. TCA - 12 -2 and TCB -7.1 was cloned into vector pIRES2 - AcGFP1 firstly, then subcloned into vector pDC315. The recombinant plasmid pDC315 -TCA- 12 -2 -TCB -7.1 was verified by restriction enzyme digestion and sequencing, the positive recombinant plasmid was transferred into 293 cells using Lipofectamine 2000. The expressions of gene TCA - 12 -2 and TCB -7.1 were identified by RT - PCR and flow cytometry. RESULTS： Both TCA - 12 -2 and TCB -7.1 genes were constructed into eukaryotic expression vector pDC315 and the expressions of genes in 293 cells were detected successfully with RT - PCR and flow cytometry. CONCLUSION： The dicistronic expression vector pDC315 - TCA - 12 - 2 - TCB - 7.1 is successfully constructed and expressed.

关 键 词：受体 抗原 T细胞 载体构建 基因表达 

分 类 号：R512.603[医药卫生—内科学] R512.62[医药卫生—临床医学]