摘要
为了制备猪急性腹泻综合征冠状病毒(SADS-CoV)核衣壳(N)蛋白多克隆抗体,采用PCR方法扩增SADS-CoV/CN/GDGL/2017毒株N基因片段,并对N基因的序列以及N蛋白氨基酸突变位点进行分析。随后,将N基因片段克隆到原核表达载体pET-32a(+)构建重组质粒,测序验证后转化至大肠杆菌BL21(DE3)感受态细胞中进行原核表达,用Ni-NTA亲和层析柱纯化N蛋白,通过免疫BALB/c小鼠获得多克隆抗体。结果表明,扩增的N基因片段和所构建的重组质粒正确无误,成功诱导表达出可溶性重组蛋白SADS-CoV-N,分子质量约为67 ku,最佳诱导时间为6 h。Western-blot以及间接免疫荧光试验结果表明,该多克隆抗体能特异性检测SADS-CoV,证明重组蛋白有良好的免疫原性。本研究为后续建立SADS-CoV快速诊断方法及该病毒致病机制的深入研究奠定基础。
To prepare the polyclonal antibody against the N protein of swine acute diarrhea syndrome coronavirus(SADS-Co V),the N gene fragment of SADS-Co V/CN/GDGL/2017 was amplified by PCR and the sequence and amino acid mutations of N gene were analyzed.Then the PCR product was cloned into the pET-32 a(+)expression vector and expressed in Escherichia coli BL21(DE3)after sequencing.Polyclonal antibody was obtained from immunizing BALB/c mice with recombinant N protein purified by Ni-NTA affinity chromatography column.The results showed that the amplified N gene fragment sequence and the constructed recombinant plasmid were correct.The recombinant protein SADS-Co V-N with 67 ku in molecular weight was successfully induced and the optimal induction time was 6 h.Western-blot and indirect immunofluorescence assay results showed that the polyclonal antibody could specifically detect SADS-Co V,indicating the recombinant protein N had good immunogenicity.This study can lay the foundation for the establishment of a rapid diagnostic method for SADS-CoV and further study on the pathogenesis of this virus.
作者
周芝海
谭耀荣
郑瑶瑶
曾繁文
麦凯杰
马静云
ZHOU Zhi-hai;TAN Yao-rong;ZHENG Yao-yao;ZENG Fan-wen;MAI Kai-jie;MA Jing-yun(College of Animal Science,South China Agricultural University,Guangzhou 510642,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2019年第9期1179-1186,共8页
Chinese Veterinary Science
基金
广州市科技计划项目（201904010433）
关键词
猪急性腹泻综合征冠状病毒
N蛋白
原核表达
多克隆抗体
swine acute diarrhea syndrome coronavirus
nucleocapsid protein
prokaryotic expression
polyclonal antibody
