鸡MHCⅠα和β2m链基因真核表达载体的构建及其在293T细胞中的表达  

Construction of Eukaryotic Expression Vector of Chicken MHCⅠα and β_2m Genes and Its Expression in 293T Cells

作　　者：戴银[1] 胡晓苗[1] 赵瑞宏[1] 侯宏艳[1] 沈学怀[1] 潘孝成[1] 周学利[1] 张丹俊[1] 

机构地区：[1]安徽省农业科学院畜牧兽医研究所,合肥230031

出　　处：《中国畜牧兽医》2016年第8期1938-1943,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(31302044);国家蛋鸡产业技术体系项目(CARS-41);安徽省科技攻关项目(11010302119)

摘　　要：为进一步研究主要组织相容性复合体Ⅰ类(major histocompatibility complex,MHCⅠ)分子在免疫应答中的作用机制,试验经PCR克隆获得MHCⅠα和β_2m链基因,随后插入带有荧光标签蛋白的真核表达载体,成功构建了重组质粒pEGFP-MHCⅠα和pmCherry-MHCⅠβ_2m,并通过脂质体法转染293T真核表达细胞。荧光显微镜观察可见,MHCⅠα和β_2m分子主要分布于真核细胞的内膜系统,改变了与之融合的荧光蛋白在细胞内的定位。此外,Western blotting检测可见大小约68.3和41.3ku的目的蛋白反应带,表明重组质粒能够在293T细胞中顺利表达,且表达蛋白与鸡MHCⅠ类分子的相应抗体可发生特异性结合反应,具有良好的免疫学活性。To explore the mechanism of MHCⅠ molecule in immune response,chicken MHCⅠα and β2m genes were cloned by PCR.Then the fragments were inserted into the eukaryotic expression vector with fluorescent protein,and the recombinant plasmids pEGFP-MHCⅠαand pmCherry-MHCⅠβ2m were constructed.The recombinant plasmids were transfected into 293 Tcell with lipofectin reagent.The gene products of recombinant plasmids were mainly located to endomembrane system of the cells by fluorescence microscopy,and changed the intracellular localization of the fusion with the fluorescent protein.Moreover,the positive reactions were observed by the method of Western blotting,and the proteins had the molecular weight of 68.3 and 41.3ku,respectively,in accord with the target proteins.The results showed that the recombinant plasmids were expressed in 293 Tcells with a good immunological activity,and the proteins had the binding reaction with specific antibodies.

关 键 词：鸡MHCⅠα基因 鸡MHCⅠβ2m链基因 真核表达载体 293T细胞 

分 类 号：Q786[生物学—分子生物学]