TCRγV_1/pcDNA3重组质粒的构建

Construction of T-cell Recepptor γV_1 Rearrangement Gene Expression Ve ctor in Eucaryotic Cells

目的:构建含TCRγV1重排基因的真核表达质粒。方法:从人淋巴瘤Jurkat细胞中提取mRNA,用RT-PCR法扩增含BamH与Hind酶切位点TCRγV1基因序列,对pcDNA3载体及TCRγV1基因PCR产物经双酶切,用连接酶将两者连接并转化到大肠杆菌DH5a,对重组质粒经序列测定,称pcDNA3/TCRγV1。结果:电泳获得333bp的TCRγV1预期条带。结论:该条带测序证实为TCRγV1基因序列。

Objective：To construct the eucaryotic exprcssion vector of TCRγV1 rearrangement gene. Methods：The mRNA of TCRγV1 was extracted from human Jurkat cells.-TCRγV1 rearrangement gene containing Barn H Ⅰ and Hind Ⅲ endoenzyme sites was obtained using RT-PCR method. The expressing plsasmid pcDNA3 and TCRγV1 gene were cleaved with 2 restriction endonuclease BaamH Ⅰ and Hind Ⅲ：Both fragments were connected using ligase and transformed into bacteria DHSa;Clonies were screened and the recombinant was sequenced for identification. Result：The expected 333bp band of TCRγV1 was obtained by eletrophoresis. Conclusion：The band is confirmed TCR V1 gene array byallgnment determination.