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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。
Objective To construct eukaryotic expression vectors TCR Vα-pIRES2-EGFP and TCR Vβ-pIRES2-EGFP carrying TCR Vα and TCR Vβ gene fragments specific to the associated antigen of diffuse large B non-Hodgkin’s lymphoma (DLBL) Transfection of Raji and Jurkat cells. Methods Previous studies found that clonal proliferating T cell receptor (TCR) Vα6 and Vβ13 subfamily T cells were found in peripheral blood mononuclear cells of one patient with DLBL. Based on this, TCR Vα6 and TCR Vβ13 genes were amplified by RT-PCR After sequencing, the eukaryotic expression vector pIRES2-EGFP with green fluorescent protein (EGFP) was cloned into the vector. The recombinant plasmids were identified by restriction endonuclease analysis and nucleic acid sequence analysis. The recombinant plasmids were identified as TCR Vα6-pIRES2-EGFP and TCR Vβ13-pIRES2- Were transfected into Raji cells and Jurkat cells respectively or co-transfected by nucleofector technique. After 24 h, the transient expression of EGFP was observed by laser scanning confocal microscopy. The expression of TCR Vα6 And TCR Vβ13 gene expression, Western blot detection of EGFP protein expression. Results The full-length sequences of TCR Vα6 and TCR Vβ13 were obtained from DLBL patients. The recombinant plasmids of TCR Vα6-pIRES2-EGFP and TCR Vβ13-pIRES2-EGFP were confirmed by restriction enzyme digestion and DNA sequencing. After 24 h of transfection, EGFP expression was observed under a confocal microscope, and more than 40% of the cells emitted green fluorescence. Fluorescence was similar in the transfected and cotransfected cells alone. Real-time quantitative PCR showed that both TCR Vα6 and TCR Vβ13 gene expression, cotransfection group 2 gene expression level slightly lower than the single transfected group; Western blot in transfected and cotransfected group showed EGFP protein expression, two groups of protein hybridization band intensity similar . Conclusion DLBL-specific eukaryotic expression plasmids of TCR Vα6-pIRES2-EGFP and TCR Vβ13-pIRES2-EGFP were successfully constructed and transfected into the cells simultaneously, and they were co-expressed in vitro.