水牛α1、α2、α3干扰素基因的克隆、分析及表达  

Cloning, sequence analysis and expressing of interferon alpha1, alpha2, alpha3 genes in buffalo

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 Xiaofeng;XIE Zhixun;FAN Qing;XIE Zhiqin;XIE Liji;HUANG Jiaoling;ZHANG Yanfan;ZENG Tingting;WANG Sheng;LUO Sisi;LI Meng;DENG Xianwen(Guangxi Key Laboratory of Veterinary Biotechnology,Guangxi Veterinary Research Institute,Nanning 530001,China)

机构地区：[1]广西兽医研究所广西兽医生物技术重点实验室,南宁530001

出　　处：《黑龙江畜牧兽医》2021年第4期131-137,141,共8页Heilongjiang Animal Science And veterinary Medicine

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

摘　　要：为了给体外研究水牛α1、α2、α3干扰素(interferon, IFN)的抗病毒作用提供试验材料,试验采用PCR技术克隆水牛IFN-α1、IFN-α2、IFN-α3编码区序列(CDS),分别采用DNAMAN、 MEGA7.0软件进行抗原肽段的预测、进化树的绘制,构建原核表达载体pET-32a-IFN-α1、pET-32a-IFN-α2、pET-32a-IFN-α3,将以上载体转入至BL21(DE3)感受态细胞中,用1.0 mmol/L IPTG分别在37,25℃下对重组菌诱导6 h,通过SDS-PAGE、Western-blot分析外源基因在大肠杆菌的表达情况。结果表明:水牛IFN-α1、IFN-α2、IFN-α3编码区序列全长分别为570,588,555 bp,分别编码189,195,184个氨基酸。水牛IFN-α1、IFN-α3与山羊IFN-α、牛IFN-αclassⅠ亲缘关系较近,与鸡IFN-α、爪蟾Ⅰ型IFN亲缘关系较远;水牛IFN-α2与牛IFN-αⅡ最近。在37℃下诱导表达,水牛IFN-α1、IFN-α2、IFN-α3重组蛋白主要以不可溶性蛋白形式存在;当其他条件不变,把诱导温度降到25℃时,重组蛋白主要以可溶性蛋白形式存在,经Western-blot鉴定为目的蛋白。说明水牛IFN-α1、IFN-α2、IFN-α3蛋白在BL21(DE3)大肠杆菌中表达成功。In order to provide experiment materials for the in vitro study of the antiviral effect of IFN-α1,IFN-α2 and IFN-α3 in buffalo, the coding sequences(CDS) of IFN-α1,IFN-α2 and IFN-α3 were cloned by PCR technology. Antigen peptides were predicted and evolutionary tree were mapped by DNAMAN and MEGA7.0 software, respectively. Prokaryotic expression vector containing IFN-α1,IFN-α2,IFN-α3 were reconstructed. The expression vectors were transferred into BL21(DE3) competent cells, and 1.0 mmol/L IPTG was used to induce the recombinant bacteria at 37 and 25 ℃ for 6 h, respectively. The expression of exogenous genes in E.coli were detected by SDS-PAGE and Western-blot. The results showed that total length of IFN-α1, IFN-α2, IFN-α3 CDS were 570, 588 and 555 bp, encoding 189, 195 and 184 amino acid residues, respectively. Buffalo IFN-α1, IFN-α3 had close correlation with Capra hircus IFN-α and Bos taurus IFN-α classⅠ, but had minor correlation with Gallus IFN-α and Xenopus laevis typeⅠIFN. Buffalo IFN-α2 had the closest correlation with Bos taurus IFN-α class Ⅱ. The recombinant proteins of IFN-α1, IFN-α2 and IFN-α3 in buffalo were mainly in the form of insoluble protein at 37 ℃, but these proteins got soluble when the temperature changed to 25 ℃. When other conditions remained unchanged and the induced temperature was reduced to 25 ℃, the recombinant protein mainly existed in the form of soluble protein and was identified as the target protein by Western-blot. These results indicated that the proteins of buffalo IFN-α1,IFN-α2 and IFN-α3 were successfully expressed in E.coli BL21(DE3).

关 键 词：水牛 干扰素 基因克隆 编码区序列 原核表达 

分 类 号：S852.43[农业科学—基础兽医学]