摘要
目的构建EBV特异性细胞毒T细胞(CTL)相关的TCR Vα-pIRES-TCR Vβ真核双表达质粒。方法通过RT-PCR和基因扫描技术分析EBV特异性CTL克隆的T细胞受体(TCR)Vα和Vβ谱系表达情况及T细胞克隆性,确定其优势表达的TCR Vα15和TCR Vβ1亚家族基因的核苷酸序列(包括CDR3互补决定区),PCR扩增出TCR Vα15和TCR Vβ1基因后,分别定向克隆入真核双表达载体pIRES,构建双顺反子重组质粒TCR Vα-pIRES-TCR Vβ,转基因技术将其转染A549细胞和Molt4细胞,通过RT-PCR、实时定量PCR、流式细胞术和蛋白印迹分析检测TCR Vα15和TCR Vβ1基因的表达。结果酶切分析和核酸序列测定证实重组质粒构建正确,转染重组质粒的细胞能够表达TCR Vα15和TCR Vβ1的mRNA及蛋白。结论成功构建了EBV特异性的TCR Vα-pIRES-TCR Vβ双顺反子真核表达质粒,并在体外实现了共表达,为进一步的实验研究奠定了基础。
Objective To construct the bicistronic eukaryotic expression plasmid TCR Vα-pIRES-TCR Vβ of EBV-specific cytotoxic T cells （CTL）. Methods The RT-PCR was used to observe the expression of T cell receptor （TCR） Vα and Vβ repertoire T cells of the EBV- specific CTL clone, and the PCR products were analyzed by genescan to evaluate T cell clonality. Gene sequence of oligoclonal expansion of TCR Vα15 and Vβ1 repertoire （including complementarity determining region 3, CDR3） were analyzed by sequencing analysis. The fragments of TCR Vα15 gene and TCR Vβ1 gene amplified by PCR were cloned into the co-expression vector pIRES. The recombined plasmid was transfected into A.549 cells and Molt4 cells using lipofectamine and the expressions of TCR Vα15 and TCR Vβ1 were detected by RT-PCR, real-time PCR, flow cytometry, and western blot analysis. Results The recombined plasmid was analyzed and verified by restriction enzyme digestion and nucleotide sequencing. The bicistronic recombinant eukaryotic expression plasmid, which could express both mRNA and protein of TCR Vα15 and TCR Vβ1 in vitro, was corrected. Conclusion The bicistronic eukaryotic expression plasmid TCR Vα-pIRES-TCR Vβ of EBV-specific CTL is constructed successfully and can co-express TCR Vα15 gene and TCR Vβ1 gene in vitro, which lays a foundation for further study.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2008年第4期412-416,共5页
Immunological Journal
基金
国家高技术研究发展计划(2006AA02Z114)
广东省自然科学基金面上项目(06025213)
血液病学国务院侨办重点专科基金(51205002)
关键词
EBV
转染
细胞毒T细胞
双顺反子
Epstein-barr virus
Transfection
Cytotoxic T cells
Bicistron
