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

doi:10.16431/j.cnki.1671-7236.2016.08.002

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (8): 1938-1943.doi: 10.16431/j.cnki.1671-7236.2016.08.002

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

戴银, 胡晓苗, 赵瑞宏, 侯宏艳, 沈学怀, 潘孝成, 周学利, 张丹俊

安徽省农业科学院畜牧兽医研究所, 合肥 230031

收稿日期:2016-01-08 出版日期:2016-08-20 发布日期:2016-08-23
通讯作者: 胡晓苗, 张丹俊 E-mail:huxiaomiao66@163.com;zhangdj694@sina.com
作者简介:戴银(1980-),女,安徽蒙城人,博士,助理研究员,研究方向:兽医微生物与免疫学,E-mail:daiyin2020@163.com
基金资助:
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）

Construction of Eukaryotic Expression Vector of Chicken MHCⅠα and β₂m Genes and Its Expression in 293T Cells

DAI Yin, HU Xiao-miao, ZHAO Rui-hong, HOU Hong-yan, SHEN Xue-huai, PAN Xiao-cheng, ZHOU Xue-li, ZHANG Dan-jun

Institute of Animal Husbandry and Veterinary Science, Anhui Academy of Agricultural Science, Hefei 230031, China

Received:2016-01-08 Online:2016-08-20 Published:2016-08-23

摘要/Abstract

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β₂m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-MHCⅠβ₂m，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β₂m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41.3 ku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。

关键词: 鸡MHCⅠα 基因; 鸡MHCⅠβ ₂m链基因; 真核表达载体; 293T细胞

Abstract:

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-MHCⅠα and pmCherry-MHCⅠβ₂m were constructed.The recombinant plasmids were transfected into 293T cell 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 41.3 ku,respectively,in accord with the target proteins.The results showed that the recombinant plasmids were expressed in 293T cells with a good immunological activity,and the proteins had the binding reaction with specific antibodies.

Key words: chicken MHCⅠα gene; chicken MHCⅠβ₂m gene; eukaryotic expression vector; 293T cell

中图分类号:

Q786

戴银, 胡晓苗, 赵瑞宏, 侯宏艳, 沈学怀, 潘孝成, 周学利, 张丹俊. 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[J]. 《中国畜牧兽医》, 2016, 43(8): 1938-1943.

DAI Yin, HU Xiao-miao, ZHAO Rui-hong, HOU Hong-yan, SHEN Xue-huai, PAN Xiao-cheng, ZHOU Xue-li, ZHANG Dan-jun. Construction of Eukaryotic Expression Vector of Chicken MHCⅠα and β₂m Genes and Its Expression in 293T Cells[J]. , 2016, 43(8): 1938-1943.

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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 β₂m Genes and Its Expression in 293T Cells

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