猪瘟病毒C株特异的TCR Vα5和Vβ6真核表达载体的构建及共转染研究

Construction and Cotransfection of Eukaryotic Expression Plasmids TCR Vα5 and Vβ6 Specific for CSFV-C Strain

本实验室前期研究发现猪瘟病毒C株特异的TCR Vα5和TCR Vβ6基因家族,为进一步研究其体外表达情况,应用RT-PCR从猪外周血单个核细胞中扩增其全长基因序列,并构建TCR Vα5-pIRES2-EGFP和TCR Vβ6-pIRES2-EGFP重组质粒,将其转染293T细胞。结果显示,TCR Vα5和TCR Vβ6全长基因分别为816bp和945bp,双酶切和核酸序列测定证实TCR Vα5-pIRES2-EGFP和TCR Vβ6-pIRES2-EGFP重组质粒构建正确;转染293T细胞24h后荧光显微镜下可以看到70%以上的细胞表达EGFP蛋白。此外,实时荧光定量PCR可检测到TCR Vα5和TCR Vβ6基因的表达,单独转染组和共同转染组Western blot均可检测到EGFP蛋白的表达,且蛋白杂交带强度相似。研究结果表明,CSFV-C株特异性的TCR Vα5-pIRES2-EGFP和TCR Vβ6-pIRES2-EGFP真核表达质粒可同时转染到293T细胞中,实现体外共表达。

Previously,we detected TCR Vα5 and TCR Vβ6 gene families specific for CSFV-C strain. In this study,to further observe the expression level in vitro, RT-PCR technique was used to amplify the full-length sequences of TCR Vα5 and TCR Vβ6, and recombinant plasmids of TCR Vα5-plRES2-EGFP and TCR Vβ6-pIRES2-EGFP were also constructed and co-transfected into 293T cells to observe the expression of CSFV-C specific αβTCR gene. In result,the TCR Vα5 and TCR Vβ6 sequences were 816 bp and 945 bp, respectively; the sequences of TCR Vα5-pIRES2-EGFP and TCR Vβ6-pIERSZ-EGFP recombinant plasmids were confirmed to be correct by restriction enzyme digestion analysis and sequencing. Green fluorescence was observed with at least 70% transfected cells after 24 h post-transfection. Besides, the expression of TCR Vα5 and TCR Vβ6 were detected through real-time PCR and the EGFP protein was also verified by Western blot and the EGFP expression level was the same between two groups. These results indicated that the eukaryotic expression vector of TCR Vα5-pIRES2-EGFP and TCR Vβ6-pIRES2-EGFP specific for CSFV-C strain were constructed successfully and can be co-transfected into the 293T cells,realizing co-expression of TCR Vα5 and TCR Vβ6 gene in vitro.