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目的:制备弓形虫微线体蛋白2胞质尾段(MIC2C)蛋白片段及其多克隆抗体。方法:以弓形虫cDNA文库为模板,PCR扩增135bp MIC2C基因片段,构建MIC2C/pGEX-4T-1原核表达系统;IPTG诱导表达GST-MIC2C融合蛋白;用纯化的融合蛋白加免疫佐剂背部皮内注射免疫新西兰兔,制备多克隆抗体,亲和层析纯化并分析抗体的效价。结果:构建了MIC2C原核表达系统,表达并纯化了GST-MIC2C融合蛋白,蛋白的浓度为3.07mg/ml;获得了抗该蛋白的兔源性抗血清,纯化后的多克隆抗体效价为1:16 000。结论:在体外制备并纯化了GST-MIC2C融合蛋白及其多克隆抗体,为后续弓形虫入侵机制的研究奠定了基础。
Objective: To prepare the fragment of cytoplasmic tail (MIC2C) of microtuber protein 2 of Toxoplasma gondii and its polyclonal antibody. Methods: The Toxoplasma gondii cDNA library was used as a template to amplify the 135bp MIC2C gene fragment and construct the prokaryotic expression system MIC2C / pGEX-4T-1. IPTG induced the expression of GST-MIC2C fusion protein. The purified fusion protein plus adjuvant New Zealand rabbits were immunized intramuscularly to prepare polyclonal antibodies. The antibody titers were purified and analyzed by affinity chromatography. Results: The prokaryotic expression system of MIC2C was constructed and the GST-MIC2C fusion protein was expressed and purified. The concentration of the protein was 3.07 mg / ml. The rabbit antiserum against this protein was obtained. The titer of purified polyclonal antibody was 1 : 16 000. CONCLUSION: The GST-MIC2C fusion protein and its polyclonal antibody were prepared and purified in vitro, which laid the foundation for further study on the invasion mechanism of Toxoplasma gondii.