狂犬病病毒CVS-11毒株基质蛋白鼠源和兔源多克隆抗体的制备、鉴定及比较
Preparation,identification and comparison of murine and rabbit polyclonal antibodies against matrix protein of rabies virus strain CVS-11
牟韶璐 1范家琛 1崔灿 1张曦 1许运斌1
作者信息
- 1. 遵义医科大学贵州省普通高等学校传染病与生物安全特色重点实验室,贵州遵义 563000
- 折叠
摘要
目的 制备狂犬病病毒(rabies virus,RV)基质蛋白(M)鼠源和兔源多克隆抗体,并比较其反应原性.方法 以RV CVS-11毒株感染细胞后的cDNA为模板构建原核表达载体pET-28a-M,转化E.coli BL21(DE3),IPTG诱导表达M蛋白,经镍柱亲和层析、透析复性后,免疫雌性BALB/c小鼠和雌性新西兰大白兔,取全血,分离血清,ELISA法检测鼠源和兔源多克隆抗体效价,Western blot法、间接免疫荧光试验(immunofluorescence assay,IFA)、免疫沉淀(immunoprecipitation,IP)法检测鼠源和兔源多克隆抗体反应原性.结果 质粒pET-28a-M经测序鉴定证明构建正确;制备的鼠源和兔源多克隆抗体效价分别为1∶100和1∶256 000,与不同RV毒株均具有反应原性.结论 成功制备了 M蛋白鼠源和兔源多克隆抗体,为探讨M蛋白与宿主蛋白相互作用的关系以及研究RV的致病机制提供了重要的生物学工具.
Abstract
Objective To prepare murine and rabbit polyclonal antibodies against rabies virus(RV)matrix(M)protein and compare their reactivity.Methods The prokaryotic expression vector pET-28a-M was constructed by using the cDNA of cells infected with RV CVS-11 strain as template,then transformed into E.coli BL21(DE3),and the induced by IPTG to express M protein.After nickel column affinity chromatography and dialysis renaturation,female BALB/c mice and New Zealand white rabbits were immunized with the M protein,and the whole blood was taken to separate the serum.The titers of the murine and rabbit polyclonal antibodies were detected by ELISA,and the reactivity was measured by Western blot,indirect immunofluorescence assay(IFA)and immunoprecipitation(IP).Results The plasmid pET-28a-M was constructed correctly as identified by sequencing.The titers of murine and rabbit polyclonal antibodies were 1∶100 and 1∶256 000 respectively,and the polyclonal antibodies had reactivity with different RV strains.Conclusion The murine and rabbit polyclonal antibodies against M protein were successfully prepared,which provides important biological tools for exploring the interaction between M protein and host protein as well as studying the pathogenesis of RV.
关键词
狂犬病病毒/基质蛋白/原核表达载体/鼠源/兔源/多克隆抗体Key words
Rabies virus(RV)/Matrix protein(M protein)/Prokaryotic expression vector/Murine source/Rabbit source/Polyclonal antibody引用本文复制引用
基金项目
国家自然科学基金(31860705)
贵州省科技计划(课题)(黔科合基础[2020]1Y061)
出版年
2024
NETL
NSTL
万方数据