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∶256000,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。
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 1:256000respectively,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.
作者
牟韶璐
范家琛
崔灿
张曦
许运斌
MU Shaolu;FAN Jiachen;CUI Can;ZHANG Xi;XU Yunbin(Key Laboratory of Infectious&Biosafety,Department of Education Guizhou,Zunyi Medical University,Zunyi 563000,Guizhou Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2024年第3期310-315,共6页
Chinese Journal of Biologicals
基金
国家自然科学基金(31860705)
贵州省科技计划项目(课题)(黔科合基础[2020]1Y061)。
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
Rabies virus(RV)
Matrix protein(M protein)
Prokaryotic expression vector
Murine source
Rabbit source
Polyclonal antibody
