藏猪IFN-λ3成熟肽基因克隆及原核表达、抗病毒效价测定  

Cloning and prokaryotic expression of IFN-λ3 mature peptide from Tibetan pig and determination of antiviral potency

作　　者：李原野 殷玥[1] 陈瑛琪[1] 张继宗 杨晓宇[1] 王佳煜 朱玲[1,2] 徐志文[1,2] LI Yuanye;YIN Yue;CHEN Yingqi;ZHANG Jizong;YANG Xiaoyu;WANG Jiayu;ZHU Ling;XU Zhiwen(School of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Key Laboratory of Animal Disease and Human Health of Sichuan Province,Sichuan Agricultural University,Chengdu 611130,China)

机构地区：[1]四川农业大学动物医学院,成都611130 [2]四川农业大学动物疫病与人类健康四川省重点实验室,成都611130

出　　处：《东北农业大学学报》2018年第10期62-69,共8页Journal of Northeast Agricultural University

基　　金：四川省科技计划项目(2017NZ0038);国家"十二五"科技计划项目(2015BAD12B04-2.3)

摘　　要：为探究藏猪Ⅲ型干扰素抗病毒作用,以藏猪肠道和肺脏核酸为模板,采用RT-PCR扩增克隆IFN-λ3基因(ZPoIFN-λ3),经测序鉴定后构建原核表达载体pET-32a(+)-mZPoIFN-λ3,并于大肠杆菌BL21(DE3)中表达。通过优化诱导时间、IPTG浓度,利用镍柱亲和层析纯化表达蛋白,并采用SDS-PAGE验证。纯化蛋白经透析复性浓缩,以此为免疫原制备鼠抗ZPoIFN-λ3多克隆抗体,采用Western Blot鉴定。通过细胞病变抑制法测定表达蛋白在MDBK/VSV系统的抗病毒活性。结果表明,成功克隆藏猪IFN-λ3成熟肽基因,经分析发现藏猪IFN-λ3核酸序列与野猪核酸同源性为100%。成功构建藏猪IFN-λ3原核重组表达质粒,并成功表达大小为34 ku蛋白,蛋白纯化后SDS-PAGE显示为单一条带,Western Blot鉴定结果显示鼠抗ZPoIFN-λ3多克隆抗体具有良好免疫原性。p ET-32a(+)-mPoIFN-λ3表达蛋白在MDBK/VSV系统抗病毒效价为10×24.5U·0.1 mL-1,比活力为2×103U·mg-1。研究首次克隆藏猪IFN-λ3基因并原核表达,通过研究其抗病毒作用,为进一步研究藏猪IFN-λ3生物学特性和相关基因重组药物生物学功能奠定基础,为临床新药物研发提供新方向。To explore the antiviral effect of Tibetan pig type III interferon,the IFN-λ3 gene(ZPoIFN-λ3)was amplified and cloned by RT-PCR using Tibetan porcine intestinal and lung nucleic acids as templates.The expression vector pET-32a(+)-mZPoIFN-λ3 was expressed in E.coli BL21(DE3).The expression protein was purified by nickel column affinity chromatography by optimizing the induction time and IPTG concentration and verified by SDS-PAGE.The purified protein was re-concentrated by dialysis,and a mouse anti-ZPoIFN-λ3 polyclonal antibody was prepared as an immunogen and identified by Western Blot.The antiviral activity of the expressed protein in the MDBK/VSV system was determined by cytopathic inhibition.The results showed that the porcine IFN-λ3 mature peptide gene was successfully cloned,and the homology of Tibetan pig IFN-λ3 nucleic acid sequence with wild boar nucleic acid was found to be 100%.The recombinant IFN-λ3 prokaryotic expression plasmid was constructed and successfully expressed as 34 ku protein.After purification,SDS-PAGE showed a single band.Western-Blot identification showed that the mouse anti-ZPoIFN-λ3 polyclonal antibody had good immunogenicity.The antiviral titer of pET-32a(+)-mPoIFN-λ3 expressing the protein in MDBK/VSV system was 10×24.5 U·0.1 mL^-1,and the specific activity was 2×103 U·mg^-1.The cloning and prokaryotic expression of IFN-λ3 gene in Tibetan pigs were studied for the first time.Through the study of its antiviral effect,it lays a foundation for further study on the biological characteristics of Tibetan porcine IFN-λ3 and the biological functions of related recombinant drugs,and offers new directions for new clinical drug development.

关 键 词：藏猪 IFN-λ3 克隆 原核表达 抗病毒 

分 类 号：S828[农业科学—畜牧学]