利用重组蛋白制备和鉴定分枝杆菌HSP65单克隆抗体

Preparation and identification of monoclonal antibody against a 65KDa heat shock protein from mycobacterium bovis BCG by recombination protein

目的制备针对牛分枝杆菌热休克蛋白65(HSP65)的单克隆抗体(mAb)并进行鉴定。方法以CpGODN1826/Alum为佐剂,不同的HSP65融合蛋白为免疫抗原和检测抗窄,用聚乙二醇(PEG)法制备杂交瘤细胞,ELISA检测杂交瘤细胞分泌抗体的效价和类型,Western blot分析抗体的特异性。结果获得5株稳定分泌抗HSP65蛋白的McAb,3株细胞分泌的mAb为IgG1亚类,2株为IgG2a。ELISA和Western blot结果显示,mAb能特异性结合HSP65-PSA-tag、HSP65-MUC1-tag和HSP65重组蛋白,但与Chaperon-PSA重组蛋白和宿主菌BL21的菌体裂解蛋白不发生结合。结论纯化的重组蛋白可代替的牛分枝杆菌,用于HSP65 mAb的制备和鉴定。获得的HSP65 mAb为HSP65及其重组蛋白的功能研究奠定基础。

Objective To prepare monodonal antibody （mAb） against a 65KDa heat shock protein from Mycobacterium boris BCG.Metheds The HSP65-histag protein acts as antigen mixed with CpG ODN1826 and AI （HO）3 to immunize BALB/c mice. The splenocyte derived from immunized mice of CpG ODN1826 / Alum adjuvant group were fused with NS1 myeloma cells by a routine PEG method. The titer and subtype of antibody secreted by the hybridoma cells were detected by ELISA, the specificity of antibody were detected by western blot. Results Anti-HSP65 mAb can specific react with HSP65 of the HSP65-PSA,HSP65-MUC1-tag and HSP65 protein, while can not react with Chaperon-PSA and lysate from the host bacteria E.celi. Conclusion The prepared anti-HSP65 MeAb can be used to detect HSP65 of various fusion proteins.