cblN/Zap嵌合分子的构建及其对Jurkat细胞TCRζ的下调  

Construction of cblN/Zap and its down-regulating effect on TCRζ protein in Jurkat cells

作　　者：李霞[1] 沈岚[1] 张璟[1] 刘新平[1] 刘云才 药立波[1] 

机构地区：[1]第四军医大学生物化学与分子生物学教研室,陕西西安710032 [2]LaJolla变态反应与免疫学研究所,美国加州92121

出　　处：《细胞与分子免疫学杂志》2005年第4期437-441,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家杰出青年科学基金资助项目(No.30228012)

摘　　要：目的:构建cblN/Zap嵌合分子,稳定转染Jurkat细胞后观察对细胞TCRζ的影响。方法:提取Jurkat细胞的总RNA并反转录为cDNA,以其作为模板用PCR方法扩增Zap基因的SH2片段。以含有人全长cblcDNA的质粒pEFHAcbl作为模板扩增cbl基因的N端(cblN),并在其N末端带上含24bp的flag标签。用重叠延伸PCR法,在cblN基因内部的SH2两端引入BamHI和EcoRV酶切位点并克隆入pcDNA3.1(+)中。再以Zap基因的SH2置换cblN基因的SH2,即成为pcDNA3.1(+)cblN/Zap。酶切鉴定及测序正确后,采用脂质体法稳定转染Jurkat细胞,用RTPCR和Westernblot鉴定flagcblN/Zap的表达,用Westernblot检测其对细胞TCRζ表达的影响。结果:重组质粒pcDNA3.1(+)flagcblN/Zap经酶切后可产生与理论预期长度相符的片段。且测序证实其序列正确。脂质体法稳定转染Jurkat细胞后,检测到目的基因在RNA和蛋白水平的表达。表达的cblN/Zap可下调Jurkat细胞的TCRζ。结论:重构嵌合分子cblN/Zap可下调Jurkat细胞的TCRζ。AIM: To construct the chimeric cblN/Zap molecule, and to observe its effect on TCRζ protein in pc- DNA3.1 (+)-cblN/Zab transfected Jurkat cells. METHODS: Total RNA of Jurkat cells were isolated and reversely transcribed into cDNA, which were used as templates to amplify Zap SH2 by PCR. cblN gene tagged with 24 bp flag was amplified by PCR using pEFHAcbl plasmid encoding human cbl as templates. BamH I and EcoR V restriction enzyme digestion sites were introduced into flank SH2 in pcDNA3.1(+) cblN by overlapping extension PCR. Then pcDNA3.1(+)-cblN/Zap was obtained by replacing SH2 of cblN with ZapSH2. After confirmation by enzyme digestion and sequencing, the recombined vector was stably transfected into Jurkat cells with lipofectin. The expression of flag-cblN/Zap was detected by RT-PCR and Western blot, and its effect on TCRζ protein was analyzed by Western blot. RESULTS: Restriction enzyme digestion analysis of the pcDNA3.1(+)-cblN/Zap recombinant vector showed that the expected fragments were produced and confirmed by sequencing. pcDNA3.1(+)-cblN/Zap was stably transfected into Jurkat cells and the expression of flag-cblN/Zap in the stable clones was confirmed by both RT-PCR and Western blot. The expressed cblN/Zap down-regulated TCRζ protein in Jurkat cells. CONCLUSION: Chimeric cblN/Zap is able to downregulate TCRζ in Jurkat cells.

关 键 词：CBL 重叠延伸PCR JURKAT JURKAT细胞 

分 类 号：Q78[生物学—分子生物学] R392.11[医药卫生—免疫学]