犬干扰素α1基因在大肠埃希菌中的表达及其抗病毒活性的测定  

Expression of canine interferon α1 gene in E. coli and antiviral activity of expressed product

作　　者：徐晓娟[1] 王怡飞[1] 王玉[1] 米政实[1] 黄永亮[1] 郭霄峰[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《中国生物制品学杂志》2013年第8期1084-1087,1093,共5页Chinese Journal of Biologicals

摘　　要：目的在大肠埃希菌中高效表达重组犬干扰素α1(canine interferon alpha1,CaIFNα1)基因,并对其抗病毒活性进行初步研究。方法根据大肠埃希菌密码子的偏嗜性,人工合成犬干扰素α1成熟蛋白编码基因,同时引入大肠埃希菌EcoRⅠ和XhoⅠ酶切位点,将改造后合成的CaIFNα1核苷酸序列定向克隆至原核表达载体pET-32a(+),构建重组表达质粒pET-CaIFNα1,并进行PCR及双酶切鉴定;将鉴定正确的重组表达质粒pET-CaIFNα1转化BL21(DE3),IPTG诱导表达,表达的重组融合蛋白经SDS-PAGE及Western blot鉴定,并采用MDCK/VSV微量细胞病变抑制法检测其抗VSV病毒活性。结果重组表达质粒经PCR及双酶切鉴定,证明构建正确;重组融合蛋白CaIFNα1相对分子质量约39 000,主要以包涵体形式存在,表达量约占菌体总蛋白的61.5%,可被鼠抗His单克隆抗体及鼠抗CaIFNα1多克隆抗体特异性识别,在MDCK细胞上呈现较高的抗病毒活性,为2.0×106U/ml。结论已成功改造了CaIFNα1基因,并在大肠埃希菌中实现了高效表达,表达产物具有较高的抗病毒活性。Objective To highly express recombinant canine interferon alpha1（CaIFNα1）gene in E.coli and investigate its antiviral activities.Methods According to the codon bias of E.coli,CaIFNα1 gene was modified,synthesized and cloned into prokaryotic expression vector pET-32a（＋）,in which the restriction sites of EcoR Ⅰ and Xho Ⅰ were introduced.The constructed recombinant plasmid pET-CaIFNα1 was identified by PCR and restriction analysis,then transformed to E.coli BL21（DE3）and for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot,and determined for the activity against vesicular stomatitis virus（VSV）by MDCK / VSV micro-CPE inhibition test.Results Both PCR and restriction analysis proved that recombinant plasmid pET-CaIFNα1 was constructed correctly.The expressed recombinant fusion protein CaIFNα1,with a relative molecular mass of about 39 000,mainly existed in a form of inclusion body and contained about 61.5% of total somatic protein,which was recognized specifically by mouse anti-His monoclonal antibody and mouse anti-CaIFNα1 polyclonal antibody and showed an antiviral activity of 2.0 × 106 U / ml on MDCK cells.Conclusion CaIFNα1 gene was successfully modified and highly expressed in E.coli,and the expressed product showed high antiviral activity.

关 键 词：干扰素Α 犬科 大肠埃希菌 原核细胞 基因表达 生物学活性 

分 类 号：Q511[生物学—生物化学] Q786