A型塞内卡病毒VP3蛋白的原核表达及多克隆抗体制备
Prokaryotic Expression of the VP3 Protein of Senecavirus A and Preparation of Its Polyclonal Antibodies
林铱婷 1姬康 1谭姗姗 1晋怡 1宋若琪 1马瑞一 1王颖 1牛胜 1赵宇军 1田文霞 1任建乐1
作者信息
- 1. 山西农业大学动物医学学院,山西晋中 030801
- 折叠
摘要
为制备A型塞内卡病毒(SVA)VP3蛋白多克隆抗体,采用同源重组技术将SVA VP3基因连接至原核表达载体pET-28a中,构建了重组表达质粒pET-SVA-VP3.将重组质粒转化至大肠杆菌感受态细胞BL21(DE3),以1 mmol/L的IPTG进行诱导表达并纯化表达产物,随后将纯化的VP3重组蛋白皮下多点注射新西兰白兔制备多克隆抗体,利用间接ELISA、间接免疫荧光试验(IFA)和Western blot分别对抗体效价、反应性和特异性进行鉴定.结果显示:重组VP3 蛋白以包涵体形式表达,相对分子质量约 36 kDa;制备的VP3 蛋白多克隆抗体能与VP3 重组蛋白和SVA发生特异性反应,其ELISA效价达 1:10 000 以上,Western blot和IFA效价达 1:5 000.综上,本研究成功制备了SVA VP3 多克隆抗体,其具有较高的效价和特异性,可为后续SVA致病机制及检测方法等研究奠定基础.
Abstract
In order to prepare polyclonal antibodies against VP3 protein of Senecavirus A(SVA),a recombinant expression plasmid named as pET-SVA-VP3 was constructed through cloning SVA VP3 gene into a prokaryotic expression vector known as pET-28a using homologous recombination technology,and transformed into Escherichia coli competent cell BL21(DE3)to express VP3 protein by IPTG induction at a concentration of 1 mmol/L,and the expressed protein was purified,then the purified VP3 recombinant protein was injected into New Zealand white rabbits via subcutaneous multi-point to prepare polyclonal antibodies,followed by evaluation on its titer,reactivity and specificity by indirect ELISA,indirect immunofluorescence assay(IFA)and Western blot.The results showed that the VP3 recombinant protein was expressed in a form of inclusion body with a molecular mass of about 36 kDa;the prepared antibodies could specifically react with VP3 recombinant protein and SVA,and the titer evaluated by ELISA reached up to above 1:10 000,and those by Western blot and IFA were both 1:5 000.In conclusion,SVA VP3 polyclonal antibodies were successfully prepared,which were with high titer and specificity.The study laid a foundation for future researches on the pathogenic mechanism and detection of SVA.
关键词
A型塞内卡病毒/VP3蛋白/原核表达/多克隆抗体/效价/特异性Key words
SVA/VP3 protein/prokaryotic expression/polyclonal antibody/titer/specificity引用本文复制引用
基金项目
山西省科技创新人才团队专项(202204051001022)
山西省优秀博士来晋奖励基金(SXBYKY2021036)
山西农业大学博士科研启动基金(2020BQ59)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据