摘要
本研究旨在采用杆状病毒表达系统表达并纯化寨卡病毒(Zika virus,ZIKV)NS1蛋白,并对其免疫原性进行鉴定。将ZIKV NS1基因克隆至杆状病毒载体pFastBac1中,构建重组转移载体,将其转化DH10Bac感受态细胞进行蓝白斑筛选。将鉴定正确的重组杆粒转染sf9细胞,获得重组杆状病毒,经SDSPAGE和Western-blot鉴定蛋白正确表达后,将其感染High5细胞,大量表达重组蛋白,并用镍柱亲和层析法进行纯化,将纯化后的ZIKV NS1蛋白免疫BALB/c小鼠,进行血清特异性抗体效价检测。最终结果显示,获得了纯度较高分泌表达的ZIKV NS1蛋白,并且血清特异性抗体效价实验表明,纯化的ZIKV NS1蛋白能够诱导小鼠产生特异性免疫反应,有很好的免疫原性。本研究为ZIKV疫苗和诊断试剂盒的研发奠定了重要的基础。
This study was planned to express and purify Zika virus(ZIKV)NS1 protein using baculovirus expression system and analyze its immunogenicity.ZIKV NS1 gene was cloned into p Fast Bac1 vector and the recombinant transfer vector was constructed and transformed into DH10Bac competent cells for blue-white spot screening.Then,the identified recombinant bacmid was transfected into sf9 cells to generate recombinant baculovirus.After SDS-PAGE and Western-blot identification,the recombinant baculovirus was infected with High5 cells to obtain recombinant NS1 protein in large quantities,and finally the recombinant protein was purified by nickel column affinity chromatography and immunized to BALB/c mice for serum specific antibody titer detection.The final results showed that secretory expressed ZIKV NS1 protein with high purity was obtained and the serum specific antibody titer test showed that the purified ZIKV NS1 protein could induce specific immune response in mice,with good immunogenicity.This study lays an important foundation for the development of ZIKV vaccine and diagnostic kit.
作者
魏思琪
张路平
张艳芳
叶静
曹胜波
陈政
WEI Si-qi;ZHANG Lu-ping;ZHANG Yan-fang;YE Jing;CAO Sheng-bo;CHEN Zheng(College of Veterinary Medicine,Huazhong Agricultural University,Wuhan A30070,China;State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2020年第3期346-352,共7页
Chinese Veterinary Science
基金
“十三五”国家重点研发计划“边境地区外来动物疫病阻断及防控体系研究”(2017YFD0501803)
“潜在入侵的畜禽疫病监测与预警技术研究”(2016YFD0501102)。
