间接免疫荧光法检测鼠疫杆菌F1抗体

An experimental study on indirect immunofluorescent assay of Y. pestis F1 antibody detection with eukaryotic expressed antigen

目的构建鼠疫杆菌F1抗原结构基因Caf1的重组载体pCDNA3.1-hisB-Caf1,在NIH3T3细胞中稳定表达,并用表达F1抗原的细胞株作为诊断抗原,探讨间接免疫荧光法(IFA)检测鼠血清中鼠疫杆菌F1抗体的可行性。方法将caf1基因克隆到质粒pCDNA3.1-hisB的多克隆位点上,构建重组载体pCDNA3.1-hisB-caf1,重组载体经脂质体转染至NIH3T3细胞中,G418筛选获得抗性细胞株,并以此细胞株为诊断抗原,IFA检测鼠血清中的F1抗体。结果经IFA证实,pCDNA3.1-hisB-caf1能在NIH3T3细胞中稳定表达,并能与1例经ELISA检测为F1抗体阳性的鼠血清发生特异性反应。结论NIH3T3细胞稳定表达的F1抗原可作为IFA中的侯选抗原,检测鼠血清中的F1抗体。

Objective To establish an indirect immunofluorescent assay（IFA）of Y.pestis F1 antibody detection in rat serum with expressed F1 antigen.Methods pCDNA3.1-hisB-caf1 was reconstructed by gene clone and recombination techniques.The vector was then transfected into NH3T3 cells with lipofectamine.The positive cell clones were selected with G418.The cells stably expressing F1 antigen were used to detect F1 antibody in rat serum with IFA.Results The recombinant vector expressed the F1 protein stably in NH3T3 cells.The purified F1 protein could react specifically with rat serum,which was confirmed by ELISA.Conclusion The F1 protein expressed in NH3T3 cells could be a valuable candidate of antigen for detecting F1 antibody in rat.