【摘要】 目的构建携带弥漫大B型非霍奇金淋巴瘤(DLBL)相关抗原特异性的TCR Vα和TCR Vβ基因片段的真核表达载体TCR Vα-pIRES2-EGFP和TCR Vβ-pIRES2-EGFP,将其共同转染Raji和Jurkat细胞。方法前期研究从1例DLBL患者外周血T细胞中发现克隆性增殖T细胞受体(TCR)Vα6和Vβ13亚家族T细胞,在此基础上利用RT-PCR扩增TCR Vα6和TCR Vβ13基因全长序列后,分别将其定向克隆入带有绿色荧光蛋白(EGFP)的真核表达载体pIRES2-EGFP,酶切和核酸序列测定分析方法鉴定重组质粒TCR Vα6-pIRES2-EGFP和TCR Vβ13-pIRES2-EGFP的正确性;利用核转染技术(nucleofector)将其分别或共同转染Raji细胞和Jurkat细胞,24 h后利用激光共聚焦显微镜观察EGFP的瞬时表达情况,48 h后利用实时定量PCR检测TCR Vα6和TCR Vβ13基因的表达情况,Western blot检测EGFP蛋白的表达情况。结果获得来自DLBL患者的TCR Vα6和TCR Vβ13基因全长序列,酶切分析和核酸序列测定证实TCR Vα6-pIRES2-EGFP和TCR Vβ13-pIRES2-EGFP重组质粒构建正确;转染24 h后,激光共聚焦显微镜下可观察到EGFP的表达,40%以上细胞发出绿色荧光,单独转染和共转染组荧光产生情况相似;实时定量PCR在单独转染和共转染组均可检测到TCR Vα6和TCR Vβ13基因的表达,共转染组2基因的表达水平稍低于单独转染组;Western blot检测在单独转染和共转染组均显示EGFP蛋白的表达,2组的蛋白杂交带强度相似。结论成功构建了DLBL特异性的TCR Vα6-pIRES2-EGFP和TCR Vβ13-pIRES2-EGFP真核表达质粒,两者可同时转染到细胞中,并实现了体外共表达。更多还原

【Abstract】 Objective To construct the eukaryotic expression plasmids TCR Vα-pIRES2-EGFP and TCR Vβ-pIRES2-EGFP specific for the diffuse large B-cell lymphoma(DLBL) associated antigen and to co-transfect the plasmids to both Raji and Jurkat cell lines.Methods Based on our previous study in which clonally expanded TCR Vα6 and TCR Vβ13 subfamily T cells were identified in a case of DLBL,the full-length TCR Vα6 and Vβ13 genes were amplified by RT-PCR and cloned into eukaryotic expression vector pIRES2-EGFP to obtain TCR Vα6-pIRES2-EGFP and TCR Vβ13-pIRES2-EGFP recombinant plasmids,respectively,and then the sequences of the recombinant plasmids were confirmed by restriction enzyme digestion analysis and sequencing.TCR Vα6-pIRES2-EGFP and TCR Vβ13-pIRES2-EGFP were transfected alone or co-transfected into both Raji cells and Jurkat cells using nucleofector technology.After 24 h,the transient expression of EGFP was observed under laser confocal microscopy(LCM).The mRNA of TCR Vα6 gene and TCR Vβ13 gene were detected by real-time PCR and the expression of EGFP protein was analyzed by Western blotting at 48 h after transfection.Results The full-length TCR Vα6 and Vβ13 genes were obtained from a case of patient with DLBL;TCR Vα6/Vβ13-pIRES2-EGFP recombinant plasmids were verified by restriction enzyme digestion and nucleotide sequencing.Green fluorescence was emitted from 40% of transfected cell under LCM 24 h post-transfection,while fluorescent emittion in the single transfection groups was similar with that in the co-transfection group.The recombinant eukaryotic expression plasmids,which could express the mRNA of TCR Vα6 and TCR Vβ13 gene as well as the EGFP protein in vitro,were conformed by real-time PCR and Western blotting 48 h post-transfection,respectively.For the mRNA expressions of TCR Vα6 and TCR Vβ13 genes,the single transfection groups were superior to the co-transfection group,while the expression level of EGFP protein was similar between two groups.Conclusion The eukaryotic expression plasmids of TCR Vα6-pIRES2-EGFP and TCR Vβ13-pIRES2-EGFP specific for DLBL are constructed successfully and could transfect simultaneously into the cells and co-express TCR Vα6 and Vβ13 gene in vitro.更多还原