鸡α干扰素基因真核表达载体pCAGGS-IFN-α的构建及其在293T细胞中的表达  

Construction of eukaryotic expression vector pCAGGS-IFN-alpha of chicken interferon α gene and its expression in 293T cells

作　　者：龚浪[1] 林宏文 崔进[1] 宋亚芬[1] 张烁[1] 邢凯祥 李梦[1] 徐成刚[1] 焦培荣[1] 廖明[1] 

机构地区：[1]华南农业大学兽医学院人兽共患病防控制剂国家地方联合工程实验室/农业部兽用疫苗创制重点实验室/广东省动物源性人兽共患病预防与控制重点实验室,广州510642 [2]内蒙古自治区通辽市扎鲁特旗农牧业局,内蒙古通辽029100

出　　处：《黑龙江畜牧兽医》2014年第8期21-23,27,268,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：科技部国际合作项目(2013DFA31940);国家肉鸡产业技术体系项目(nycytx-42-G3-03);广东省科技计划项目(2010B020307005;2012B020306003);广州市科技计划项目(2013J4500030;201300000037)

摘　　要：为了构建鸡α干扰素基因真核表达载体,试验采用RT-PCR方法从鸡胚成纤维细胞中扩增α干扰素基因序列,将扩增产物定向插入到pMD18-T克隆载体中,进行序列测定;测序正确的质粒经EcoRⅠ和SacⅠ双酶切,将目的基因片段定向克隆到真核表达载体pCAGGS中,构建重组质粒pCAGGS-IFN-α;将重组质粒转染293T细胞,使用间接免疫荧光、Western-blot等方法检测目的蛋白。结果表明:已成功扩增出582 bp大小的鸡α干扰素基因片段,测序结果与GenBank报道的序列基本一致。说明阳性重组质粒pCAGGS-IFN-α真核表达载体构建成功,能正确表达鸡α干扰素。To construct the eukaryotic expression vector of chicken interferon - alpha gene, reverse transcriptase - polymerase chain reaction （ RT - PCR） was used to amplify the interferon - alpha gene sequences from chicken embryo fibroblasts. The PCR product was directionally in- serted into pMD18 -T cloning vector for sequencing. The identified plasmid was digested with EcoR I and Sac I , and then the target gene fragment was directionally cloned into the eukaryotie expression vector pCAGGS to construct the recombinant plasmid pCAGGS - IFN -α. The recombinant plasmid pCAGGS - IFN - α was transfected into the 293T cells, and then the target protein was detected using indirect immunoflu-orescence, Western - blot, etc. The results showed that the 582 bp fragment of the chicken interferon alpha gene was successfully amplified, and the sequencing result was basically consistent with the sequence reported in GenBank. The results indicate that positive recombinant plas- mid pCAGGS - IFN - α eukaryotic expression vector is successfully constructed, and it can correctly express chicken interferon alpha.

关 键 词：鸡α干扰素 真核表达 构建 鉴定 

分 类 号：Q782[生物学—分子生物学] Q786