荣昌猪白细胞介素-2受体γ链基因克隆、表达与鉴定  

Cloning,expressiong and characterization of the Rongchang porcine IL-2 receptor γ chain gene

作　　者：廖晓丹[1] 郭万柱[1] 吴翼[1] 余光勇[1] 蒋清蓉[1] 

机构地区：[1]四川农业大学动物生物技术中心,四川雅安625014

出　　处：《中国兽医学报》2009年第12期1547-1551,共5页Chinese Journal of Veterinary Science

基　　金：国家"973"计划资助项目(2004CCA011800);教育部长江学者和创新团队发展计划资助项目(IRT0555)

摘　　要：运用RT-PCR技术从由刀豆蛋白(ConA)诱导培养的荣昌猪外周血单核淋巴细胞(PBMC)扩增出猪白细胞介素-2受体γ链基因的完整开放阅读框(ORF),长约1 107 bp,编码由368个氨基酸残基组成的相对分子质量为41 780的蛋白多肽。荣昌猪IL-2Rγ与人、猕猴、牛、犬、鼠在氨基酸水平上的同源性分别为81.6%,80.8%,85.1%,83.2%和68.8%。运用PCR技术从含荣昌猪IL-2Rγ开放阅读框序列质粒中扩增其成熟蛋白编码基因,共1 020 bp。将其定向克隆于原核表达载体pET-32a(+)后在E.coliBL21中诱导表达,SDS-PAGE结果显示表达的融合蛋白约为52 000,重组蛋白以包涵体的形式表达,表达产物约占菌体总蛋白的37.6%;Western-blotting分析表明,在相对分子质量52 000处有一条特异性的带;对γ诱导重组菌进行转录检测得到约1 020 bp的特异性片段。The complete open reading frame （ORF） of interleukin-2 receptor γ chain gene was amplified from Rongchang porcine peripheral blood lymphocyte stimulated by ConA,which includes 1 107 bp and encoding 41 780 of petide of 368 amino acids. The amino acid sequence showed 81.6%,80.8% ,85.1% ,83.2% and 68.8% similarities to that of human, macaca, bos taurus, canis familiar, mus musculus chains, respectively. The gene encoding mature IL- 2Rγ protein was amplified by PCR from the recombinant plasmid containing IL-2Rγ,whieh includes 1 020 bp in all. Then it was cloned directionally into prokaryotic expression vector pET-32a（＋）and the fusion expression was induced in E. eoli BL21. SDS-PAGE demonstrated that the fusion protein expressed in form of inclusion body is approximately 52 000, the product of expression accounts 37.6 % of the total bacterium protein. Western blotting analysis indicated that a specific electrophoresis strip was observed by detection after the recombinant bacteria was transcribed.

关 键 词：猪白细胞介素-2受体γ链基因 克隆 原核表达 

分 类 号：S328[农业科学—作物遗传育种]