巢式PCR克隆猪T细胞表面抑制性受体PD-1全长基因  

Cloning of Full-length Gene of Immune Inhibitory Receptor PD-1 on Swine T Cell Surface by Nest PCR

作　　者：朱艳平 何勇 岳锋 李鹏 韩爽 孙国鹏 张艳芳 李润芷 杨健 邢瑞林 王选年 

机构地区：[1]新乡学院生命科学技术学院,新乡学院生物技术研究中心,新乡453003 [2]河南科技学院,新乡453003

出　　处：《基因组学与应用生物学》2017年第10期4079-4084,共6页Genomics and Applied Biology

基　　金：国家自然科学基金(31272539);河南省基础与前沿技术研究(132300410353);新乡学院科技创新基金(12ZA04);2015年河南省教育技术装备和实践教育研究立项课题(No.GZS095)共同资助

摘　　要：为了获得猪T细胞表面抑制性受体PD-1的全长基因,本研究根据猪T细胞表面抑制性受体PD-1的c DNA序列,设计合成2对引物P1/P2、P3/P4,并在P1和P2的5'端分别引入Eco RⅠ和XhoⅠ酶切位点。应用巢式PCR技术从感染猪瘟病毒(CSFV)石门株的猪外周血单个核细胞中,扩增获得了大小为866 bp的基因片段。将扩增的目的基因回收、纯化,克隆至p MD-18 T克隆载体,转化宿主菌DH5α。菌液PCR和质粒PCR选择可疑阳性重克隆质粒,抽提质粒,然后用Eco RⅠ、XhoⅠ双酶切鉴定,最后进行基因序列测定。结果表明,成功构建猪PD-1全长基因的重组质粒(p MD-PD1)。In order to gain the full-length gene of immune inhibitory receptor PD-1 on swine T cell surface, two pairs of primers P1/P2, P3/P4 were designed, according to the sequence of c DNA. Eco R Ⅰ and Xho Ⅰ were respectively introduced to restriction sites in the 5＇ of P1 and P2. The full-length gene of PD-1 gene fragments were amplified by applying Nest PCR technology, which were from the peripheral blood mononuclear cells of porcine infected with CSFV in China. The DNA fragment was 866 bp. And then the PCR product was purified and sub-cloned into the cloning vector p MD-18 T. The recombinant cloning plasmid was transformed into E. coli DH5α. After identification by PCR, enzyme digestion of Eco RⅠ, Xho Ⅰ and sequencing analysis, the p MD-PD1 recombinant expression vector was constructed successfully.

关 键 词：猪 T细胞表面抑制性受体 PD-1 巢式PCR 克隆 

分 类 号：Q78[生物学—分子生物学]