摘要
A型塞内卡病毒(SVA)是一种无囊膜的单股正链小RNA病毒,是塞内卡病毒属的唯一成员,其感染后可引起仔猪的急性死亡和母猪水泡样病变。SVA编码的非结构蛋白3D在病毒增殖及拮抗宿主天然免疫反应中发挥着重要的作用。本研究旨在原核表达SVA 3D蛋白,免疫BALB/c小鼠制备多克隆抗体。以SVA RNA为模板,通过RT-PCR试验扩增3D基因,并将3D基因克隆至原核表达载体pCold-Ⅰ中,构建重组质粒3D-pCold-Ⅰ。将重组质粒转化至大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达3D蛋白,纯化该重组蛋白后免疫BALB/c小鼠制备SVA 3D蛋白的多克隆抗体。结果显示,原核表达的SVA 3D蛋白是一种可溶性蛋白,其分子质量约为55 kDa。经Western blot和IFA试验鉴定,制备的SVA 3D多克隆抗体具有良好的反应性,本研究为SVA的基础研究和应用研究提供了工具。
Senecavirus A(SVA)is the only member of genus Senecavirus,which is an envelope-free,single-stranded small RNA virus.SVA infection causes acute death in piglets and vesicular lesions in sows.The non-structural protein 3D plays an important role in virus replication and anti-host natural immune response.The purpose of this study was to express the 3D protein of SVA and prepare polyclonal antibodies.The 3D gene sequence was amplified in RT-PCR and cloned into prokaryotic expression vector pCold-Ⅰto construct recombinant plasmid 3D-pCold-Ⅰ,which was then transformed into competent cells of E.coli BL21(DE3)for protein expression with induction of IPTG.The expressed recombinant protein 3D was used to immunize mice for preparation of polyclonal antibodies.The results showed that the recombinant 3D protein was expressed in E.coli BL21 in soluble form with molecular weight of about 55 kDa.The results of Western blot and IFA showed that the polyclonal antibodies against 3D protein had good specifi city,which provided a tool for basic and applied research on SVA.
作者
程群
孙大格
翟雪滢
杨心雨
叶晨倩
叶曼青
孔宁
单同领
CHENG Qun;SUN Dage;ZHAI Xueying;YANG Xinyu;YE Chenqian;YE Manqing;KONG Ning;SHAN Tongling(Minhang Animal Disease Prevention and Control Center,Shanghai 201109,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)
出处
《中国动物传染病学报》
CAS
北大核心
2024年第5期191-195,共5页
Chinese Journal of Animal Infectious Diseases
基金
中央级公益性科研院所基本科研业务费专项(Y2022QC28)。
