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目的制备弓形虫微线体蛋白8羧基端胞质尾(MIC8 CTD)重组蛋白及其多克隆抗体。方法以弓形虫基因组为模板,PCR扩增MIC8 CTD基因片段,构建MIC8 CTD/pGEX-4T-1原核表达系统;IPTG诱导表达GST-MIC8CTD融合蛋白;用纯化的融合蛋白加免疫佐剂免疫新西兰兔,制备多克隆抗体,亲和层析纯化并分析抗体的特异性及效价。结果构建了MIC8 CTD原核表达系统,表达并纯化了GST-MIC8 CTD融合蛋白;获得了抗该蛋白的兔源性抗血清,纯化后的多克隆抗体能特异识别MIC8 CTD,ELISA测定抗体效价为1∶8 000。结论制备的GST-MIC8 CTD融合蛋白具有免疫原性,用该抗原免疫动物可获得高效价的多克隆抗体。
Objective To prepare the MIC8 CTD recombinant protein and its polyclonal antibody against Toxoplasma gondii microtubes protein 8. Methods Toxoplasma gondii genome was used as a template to amplify the MIC8 CTD gene fragment by PCR. The expression system of MIC8 CTD / pGEX-4T-1 was constructed. IPTG induced the expression of GST-MIC8CTD fusion protein. The purified fusion protein plus immune adjuvant was used to immunize New Zealand rabbits , Prepare polyclonal antibody, purify by affinity chromatography and analyze antibody specificity and potency. RESULTS: The prokaryotic expression system of MIC8 CTD was constructed, and the GST-MIC8 CTD fusion protein was expressed and purified. The rabbit antiserum against this protein was obtained. The purified polyclonal antibody specifically recognized MIC8 CTD. The titer of antibody was 1: 8 000. Conclusion The prepared GST-MIC8 CTD fusion protein is immunogenic, and the high titer polyclonal antibody can be obtained by immunizing the animal with this antigen.