新型猪α干扰素YNS突变体的构建及其抗病毒活性检测  

Construction of a novel mutant of porcine interferon-α-YNS and detection of its antiviral activity

作　　者：王朋涛 梁秀丽[2] 徐盟龙 赵福杰 闫晓光 魏战勇[1,3] WANG Pengtao;LIANG Xiuli;XU Menglong;ZHAO Fujie;YAN Xiaoguang;WEI Zhaoyong(College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;Anyang Institute of Technology,Anyang 455000,China;Key Laboratory for Animal-derived Food Safety of Henan Province,Zhengzhou 450002,China)

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]安阳工学院,河南安阳455000 [3]河南省动物性食品安全重点实验室,河南郑州450002

出　　处：《畜牧与兽医》2020年第12期116-120,共5页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划(2017YFD0501003)。

摘　　要：试验旨在获得高活性的猪α干扰素(porcine interferon alpha,PoIFN-α)。利用融合PCR方法将天然猪α干扰素基因进行定点突变,突变位点为58H(CAT)→Y(TAC)、59E(GAG)→N(AAC)及61L(CTC)→S(AGC)。将突变基因克隆到pET-32a载体上,转化至大肠杆菌Rosetta中诱导表达和鉴定,利用MTT法检测猪α干扰素对Vero细胞的抗增殖能力,利用Vero/VSV系统上测定其抗病毒活性。结果显示,重组蛋白表达形式为包涵体,蛋白分子质量约33 kDa,纯化后猪α干扰素YNS突变体蛋白的CC50为462.7μg/mL,其抗病毒活性为1.63×10^6 IU/mg,相比天然干扰素蛋白活性提高。本研究成功构建了高效猪α干扰素YNS突变体,为干扰素的临床应用提供理论基础。In order to obtain porcine interferon-αwith high activity,the natural porcine interferon-αgene was mutated by fusion PCR.The mutation sites were 58 H(CAT)→Y(TAC),59 E(GAG)→N(AAC)and 61 L(CTC)→S(AGC).The mutant gene sequence was cloned into a pet-32 a vector and was transformed into Rosetta.The results showed that the expression form of the recombinant protein was sediment,and the molecular weight of the protein was about 33 kDa.The CC50 of the purified mutant protein was 462.7μg/mL,and its antiviral activity was 1.63×10^6 IU/mg,which was higher than that of natural interferon protein.In this study,we successfully constructed the mutant of high-efficiency porcine interferon-α-YNS,which provided a theoretical basis for clinical application of interferons.

关 键 词：猪Α干扰素 基因突变 抗病毒活性 

分 类 号：S853[农业科学—临床兽医学]