摘要
寨卡病毒(ZIKV)是一种重要的人兽共患病病原。为了在原核表达系统表达ZIKV C、prM和E蛋白并制备相应多克隆抗体,本研究首先对ZIKV C、prM和E蛋白基因序列进行密码子优化,合成后克隆至pMAL-c5x载体中,重组质粒转化于E.coli ER2523,IPTG诱导表达产物用SDS-PAGE及Western blot进行鉴定并通过MBP亲和层析柱进行纯化;将纯化蛋白免疫新西兰大白兔,制备C、prM和E蛋白的兔源多克隆抗体;用IFA和Western blot检测血清抗体效价及特异性。结果显示:本研究利用原核表达系统成功表达了ZIKV C、prM和E蛋白,经过MBP亲和层析柱纯化后获得高纯度的目的蛋白;将ZIKV C、prM和E蛋白免疫新西兰大白兔后均诱导产生特异性多克隆抗体,间接免疫荧光试验表明所制备多克隆抗体均能与ZIKV结合,IFA效价分别为1:1600,1:1600和1:3200;Western blot显示制备的多克隆抗体能特异性识别ZIKV C、prM和E蛋白。该研究结果为进一步开展ZIKV相关研究奠定了基础。
In order to express Zika virus(ZIKV)C,prM and E gene using prokaryotic expression system and prepare polyclonal antibodies,we synthesized C,prM and E genes by using codon-optimized techniques,which were cloned into pMAL-c5x vectors and verified by sequencing.The recombinant plasmids were transformed into E.coli ER2523 for prokaryotic expression with IPTG induction.The resulting recombinant proteins were purified by amylose resin high flow column and examined in SDS-PAGE and Western blotting.The purified proteins were used to immunize New Zealand white rabbits to produce polyclonal antibodies.The IFA titers against C,prM and E proteins were determined to be 1:1600,1:1600 and 1:3200,respectively.The preparation of polyclonal antibodies provided a platform for further study of pathogenesis and rapid diagnosis for ZIKV infection.
作者
张经伟
王晗
刘靖
马乐
李伟
步志高
华荣虹
ZHANG Jing-wei;WANG Han;LIU Jing;MA Le;LI Wei;BU Zhi-gao;HUA Rong-hong(Harbin Veterinary Research Institute,CAAS,Harbin 150069,China)
出处
《中国动物传染病学报》
CAS
北大核心
2020年第5期42-47,共6页
Chinese Journal of Animal Infectious Diseases
基金
国家重点研发计划项目(2016YFD0500403)。
关键词
寨卡病毒
结构蛋白
多克隆抗体
Zika virus
structural protein
polyclonal antibody
