水牛白细胞介素2的原核表达与活性鉴定  

Prokaryotic Expression and Activity Identification of Recombinant Buffalo Interleukin-2

作　　者：李小凤 谢芝勋[1] 范晴[1] 谢志勤[1] 谢丽基[1] 黄娇玲 张艳芳[1] 曾婷婷[1] 王盛[1] 罗思思[1] 李孟[1] 邓显文[1] LI Xiao-feng;XIE Zhi-xun;FAN Qing;XIE Zhi-qin;XIE Li-ji;HUANG Jiao-ling;ZHANG Yan-fang;ZENG Ting-ting;WANG Sheng;LUO Si-si;LI Meng;DENG Xian-wen(Guangxi Key Laboratory of Veterinary Biotechnology,Guangxi Veterinary Research Institute,Nanning,Guangxi,530001,China)

机构地区：[1]广西兽医生物技术重点实验室,广西兽医研究所,广西南宁530001

出　　处：《动物医学进展》2021年第11期1-5,共5页Progress In Veterinary Medicine

基　　金：广西科技重大专项(桂科AA17204057);广西八桂学者专项(2019A50);国家万人计划领军人才专项(W02060083)。

摘　　要：旨在制备高纯度水牛白细胞介素2(lnterleukin,IL-2)蛋白,为研发新型抗病毒药物奠定基础。通过刀豆蛋白A(concanavalin A,ConA)刺激水牛脾脏淋巴细胞,提取细胞总RNA,利用RT-PCR扩增IL-2基因编码区序列,与表达载体pET-32a连接,构建重组表达质粒pET-32a-IL-2,转入大肠埃希氏菌BL21中,用IPTG诱导目的蛋白表达,对表达产物进行SDS-PAGE分析、纯化、Western blot鉴定及抗病毒活性评估。结果显示,水牛IL-2编码区序列全长468 bp,共编码155个氨基酸。重组IL-2蛋白主要以可溶性形式表达,表达的IL-2蛋白能与His标记抗体和山羊IL-2多克隆抗体反应,且在牛肾细胞上能有效抑制牛病毒性腹泻病毒引起的细胞病变,具有一定的抗病毒效果。结果表明,重组水牛IL-2蛋白在大肠埃希氏菌中表达量大、纯度高,反应性好,具有较强的抗病毒活性,可作为新型抗病毒药物的候选制品。The aim of this study was to get high purity recombinant protein interleukin 2 of buffalo,laying the foundation for development of new antiviral drugs.IL-2 gene coding sequence(CDS)was amplified by RT-PCR,and the total RNA from spleen lymphocytes after stimulated by ConA was linked to prokaryotic expression vetor pET-32a and transformed to competent E.coli BL21 cell,the expressed product was induced by IPTG,analyzed by SDS-PAGE,purified,identified by Western-blot,and the antiviral activity was evaluated.The results showed that CDS length of IL-2 was 468 bp,encoding 155 amino acid.The expressed IL-2 protein was maily existed in a soluble form,can react with His-tag monoclonal antibody and goat IL-2 polyclonal antibody,with good antiviral activity,effectively inhibited the cytopathic effect caused by BVDV on MDBK cells.Conclusion,the IL-2 was highly expressed in E.coli with high purity,showing good reactivity and strong antiviral activity,could be used as a candidate product of novel antiviral drugs.

关 键 词：白细胞介素2 原核表达 纯化 抗病毒活性 水牛 

分 类 号：S859.797[农业科学—临床兽医学] S858.23[农业科学—兽医学]