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.
出处
《中国兽医科学》
CAS
CSCD
北大核心
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)
关键词
猪β干扰素
真核表达
表达产物
免疫活性
porcine interferon beta
eukaryotic expression
expressed product
immune activity
