犬瘟热病毒截短P蛋白的表达及纯化  

Prokaryotic expression and purification of canine distemper virus truncated phosphoprotein

作　　者：李爽[1] 易立[1] 程悦宁[1] 曹智刚[1] 林鹏[1] 程世鹏[1] LI Shuang;YI L i;CHENG Yuening;CAO Zhigang;LIN Peng;CHENG Shipeng(Institute of Special Animal and Plant Sciences,Chinese Academy of Agricultural Sciences,Changchun 130112,China)

机构地区：[1]中国农业科学院特产研究所

出　　处：《畜牧与兽医》2019年第10期81-86,共6页Animal Husbandry & Veterinary Medicine

基　　金：吉林省特种动物生物制品科技创新中心(20170623043TC);吉林省重大科技项目人才研发团队

摘　　要：为获得犬瘟热病毒(CDV)野毒株截短磷蛋白(aa232~507),以Asia 1型CDV-PS为基础,经PCR扩增得到P(aa232~507)基因,插入原核表达载体pEASY^R-Blunt E1中,构建重组原核表达载体pEASY^R-Blunt E1-CDV-P;将重组质粒转化到BL21感受态细胞中进行原核表达并对诱导条件进行优化;超声破碎最佳条件下的诱导产物经离心后对重组蛋白进行可溶性分析,随后用镍柱对重组截短P蛋白进行纯化并用SDS-PAGE和Western blot进行鉴定。结果显示:重组P蛋白大小约37 kDa,在IPTG终浓度0.1 mmol/L,诱导时间为8 h时,表达量最高;重组P蛋白以包涵体形式存在,经SDS-PAGE和Western blot鉴定,成功纯化出重组截短P蛋白;重组P蛋白具有良好的免疫反应性。本研究成功制备出CDV-PS截短P蛋白,为进一步P蛋白抗原表位的研究和CDV抗体诊断方法的研发奠定基础。In order to obtain the canine distemper virus wild strain(CDV-PS) truncated phosphoprotein(aa232~507). This experiment was performed based on the Asia 1 CDV-PS and by PCR amplification to obtain the P gene(aa232~507). Then, the PCR product was inserted into the prokaryotic expression vector pEASY^R-Blunt E1 to construct a recombinant prokaryotic expression vector pEASY^R-Blunt E1-CDV-P. The recombinant plasmid was transferred into the BL21 competent cell for prokaryotic expression, and the induction conditions were optimized. The products induced under the optimum conditions of ultrasonic crushing were analyzed by solubility of recombinant protein after centrifugation. After that, the truncated recombinant P protein was purified by nickel column and identified by SDS-PAGE and Western blot. The results showed that the recombinant P protein was about 37 kDa;and when the final concentration of IPTG was 0.1 mmol/L and the induction time was 8 h, the highest expression of recombinant P protein was obtained in the form of inclusion body. The truncated recombinant P protein was successfully purified by SDS-PAGE and Western blot identification. The recombinant P protein had good immunoreactivity. In this study, CDV-PS truncated P protein was successfully prepared, which laid a foundation for further research on the P protein antigen epitope and for development of CDV antibody diagnosis methods.

关 键 词：犬瘟热病毒 重组P蛋白 原核表达 蛋白纯化 

分 类 号：S852.65[农业科学—基础兽医学]