鸡MxA基因的克隆及其在大肠杆菌中的表达  

Cloning of MxA Gene from Chickens and Its Expression in Escherichia coli

作　　者：陈蕾[1] 江国托 常维山[1] 

机构地区：[1]山东农业大学动物科技学院,泰安271018 [2]大连三仪生物工程研究所,大连116036

出　　处：《农业生物技术学报》2007年第2期203-206,共4页Journal of Agricultural Biotechnology

基　　金：山东省2005年重大科技专项(No.2005041005)资助。

摘　　要：MxA是由Ⅰ型干扰素诱导宿主细胞所产生的抗病毒蛋白家族的成员之一。采用RT-PCR方法从鸡胚成纤维细胞(CEF)中扩增MxA,将其克隆至载体pMD-T18中,筛选阳性克隆后回收目的片段,将其克隆入原核表达质粒pGEX-6p-1,构建其重组表达质粒pGEX-MxA,以IPTG诱导表达,经SDS-PAGE、鸡胚新城疫病毒增殖干扰实验和VSV-CEF微量细胞抑制实验进行分析、鉴定。结果表明,经RT-PCR扩增获得的MxA序列与GenBank报道的序列一致,SDS-PAGE和干扰实验证实重组质粒可以表达出相应分子量为45kD的蛋白,与GST-MxA融合蛋白分子量一致,影像分析系统分析显示表达的融合蛋白约占菌体蛋白的30%。MxA is a new kind of antivirus protein that is induced by IFN α /β. The MxA gene fragment was amplified by RT-PCR from chicken embryo fibroblast （CEF） cells and subcloned into the pMD-T18 vector and the positive clone was filtrated and the MxA gene was reclaimed and subcloned into the prokaryotic expression plasmid pGEX-6p-1. After recombinant plasmid was induced by Isopropyl 13-D-l-thiogalactopyranoside （IPTG）, the expressed protein was analyzed by SDS-PAGE, New castle disease virus （NDV） intervence experiment and Vesicular stomatitis virus （VSV）-CEF restrain experiment. Results showed that the sequence of MxA gene amplified by RT-PCR was the same as the sequence in gene map of GenBank and its protein molecular weight was 45 kD, which was the same as the fusion protein GST-MxA. And the recombinant MxA was expressed efficiently in forms of inclusion body with the yield accounting for 30% total bacteria protein by the analysis of the facility.

关 键 词：鸡 MXA 基因克隆 原核表达 生物活性 

分 类 号：S188[农业科学—农业基础科学]