猪β干扰素真核表达载体pEGFP-C1-IFN-β的构建及在CHO细胞中的表达  

Construction of a eukaryotic expression vector pEGFP-C1-IFN-β for porcine interferon beta and its expression in CHO cells

作　　者：徐雷 冯若飞[1,2] 李向茸 张海霞 谢晶莹[3] 包晓婧 马玉梅[3] 马忠仁[1,2] 

机构地区：[1]甘肃省动物细胞工程技术研究中心,甘肃兰州730030 [2]西北民族大学生物工程与技术国家民委重点实验室,甘肃兰州730030 [3]西北民族大学生命科学与工程学院,甘肃兰州730030

出　　处：《中国兽医科学》2015年第7期751-757,共7页Chinese Veterinary Science

基　　金：教育部"长江学者和创新团队发展计划"项目(IRT13091);西北民族大学研究生科研创新项目(Yxm2014187)

摘　　要：为了获得重组猪β干扰素,并实现其在CHO-K1细胞中的表达,根据GenBank中登录的IFN(JN391525.1)核苷酸序列,设计并合成目的基因的检测引物,并将合成的目的基因克隆至pEGFP-C1载体上,构建重组真核表达质粒pEGFP-C1-IFN-β,通过PCR、酶切鉴定及测序确认,将重组质粒转染到CHO-K1细胞中。经SDS-PAGE检测,获得了约25ku的表达产物,与预期的猪β干扰素蛋白的分子质量大小一致。Western-blot分析表明,表达产物与抗β干扰素阳性血清发生反应,证实表达产物具有良好的反应原性。本试验成功地在CHO-K1细胞上表达了猪β干扰素,为猪β干扰素的后续研究及生产提供了技术支持。In order to obtain recombinant porcine interferon beta and succeed to express it in CHO-K1 cells,primers for porcine IFN-β gene were designed and synthesized based on the published nucleotide se- quence of IFN-β in GenBank（JN391525.1）. The target gene was amplified and cloned into pEGFP-C1 vec- tor to construct the eukaryotic expression vector pEGFP-CI-IFN-β,and the positive recombinant plasmids were confirmed by PCR identification, double enzyme digestion analysis and sequencing. The recombinant eukaryotic expression vector was transfected into CHO-K1 cells. SDS-PAGE analysis of the cell lysate re- vealed a protein band of 25 ku,which was consistent with the expected molecular weight of porcine IFN-β. Western-blot confirmed that the expressed product was able to react with anti-IFN-β positive serum,show- ing good reactionogenicity.

关 键 词：猪β干扰素 真核表达 表达产物 免疫活性 

分 类 号：S852.43[农业科学—基础兽医学]