犬干扰素α2的原核表达及抗病毒活性分析  

PROKARYOTIC EXPRESSION AND ANTIVIRAL ANALYSIS OF CANINE INTERFERON α 2

作　　者：姚凌云[1,2] 王晶宇[2] 欧阳伟[2] 钱晶[2] 吴世妍 夏兴霞[2] 诸玉梅[2] 王晓丽[2] 潘群兴[2] 芮荣[1] 王永山[2] YAO Ling-yun;WANG Jing-yu;OUYANG Wei;QIAN Jing;WU Shi-yan;XIA Xing-xia;ZHU Yu-mei;WANG Xiao-li;PAN Qun-xing;RUI Rong;WANG Yong-shan(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agricultural,Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]南京农业大学动物医学院,南京210095 [2]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室,南京210014

出　　处：《中国动物传染病学报》2018年第2期59-63,共5页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划项目(2016YFD0501000);国家公益性行业(农业)科研专项(201303042);江苏省农业科技自主创新资金(CX(15)1065)

摘　　要：本研究将人工合成的犬干扰素α2成熟区序列插入原核表达载体p ET-28a(+)中,构建重组表达质粒p ET-28a-Ca IFN-α2;然后将该质粒转化至大肠杆菌Rosetta感受态中进行IPTG诱导表达,经SDS-PAGE和Western blot分析鉴定,分子量约为23 k Da的目的蛋白表达,表达的重组蛋白主要以包涵体的形式存在,表达量约占菌体总蛋白的52.5%。包涵体蛋白经变性、复性和纯化处理后,获得的重组Ca IFN-α2纯度为92%;用MDCK/VSV微量细胞病变抑制法检测重组蛋白的抗病毒活性为3.16×106/m L。本研究结果为进一步研制新型犬用干扰素制品奠定了物质基础。In this study,the mature encoding sequence of canine interferonα2 subtype was synthesized and inserted into the prokaryotic expression vector pET28a(+)to construct the recombinant plasmid pET-28a-CaIFN-α2.The resulting pET-28a-CaIFN-α2 was transformed into E.coli Rosetta competent cells for protein expression with induction of IPTG.The recombinant CaIFN-α2 was determined to be 23 kDa of molecular mass in SDS-PAGE and Western blot.In addition,the recombinant CaIFN-α2 mainly existed in a form of inclusion body and accounted for 52.5%of the total bacterial proteins.The recombinant CaIFN-α2 with the purity of 92%was obtained through a series of denaturation,renaturation and purification treatments.The antiviral activity of the recombinant CaIFN-α2 was determined by CPE reduction of vesicular stomatitis virus infection in MDCK cells.The results showed that the antiviral activity of the recombinant CaIFN-α2 was 3.16×106 U/mL,which laid the foundation for the development of novel canine interferon agent.

关 键 词：重组犬干扰素α2 原核表达系统 抗病毒活性 

分 类 号：S852.42[农业科学—基础兽医学]