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目的研制抗弓形虫水通道蛋白(TgAQP)的特异性多肽抗体,并应用于检测弓形虫ME49株中水通道蛋白的表达和亚细胞定位。方法根据TgAQP氨基酸序列设计B细胞抗原多肽,并将合成的多肽与KLH偶联作为免疫原免疫新西兰大耳白兔制备多克隆抗体,通过ELISA、Western blot和免疫荧光的方法对所得抗体进行鉴定。结果选定并合成抗原表位多肽,与KLH偶联作为免疫原制备多克隆抗体;经ELISA测定其多肽抗体效价达1∶40 000;Western blot表明制备的抗体能特异地识别天然弓形虫中29.9kDa处的条带,与预测的TgAQP相对分子量大小相符;免疫荧光检测到抗血清识别的蛋白定位于弓形虫胞质中。结论制备了抗弓形虫水通道蛋白多肽的多克隆抗体,为进一步研究弓形虫水通道蛋白的生物学功能和代谢特点奠定了基础。
OBJECTIVE: To develop specific polypeptide antibodies against Toxoplasma gondii aquaporin (TgAQP) and to detect the expression and subcellular localization of aquaporins in ME49 strain of Toxoplasma gondii. Methods Based on the amino acid sequence of TgAQP, the B cell antigen peptide was designed. The polyclonal antibody was prepared by immunization of New Zealand white rabbits with KLH as the immunogen. The antibodies were identified by ELISA, Western blot and immunofluorescence. Results Polypeptides of epitopes were selected and synthesized, and polyclonal antibody was prepared by conjugating with KLH as immunogen. The antibody titer of McAb reached 1: 40 000 by ELISA. The result of Western blot showed that the antibody could specifically recognize Toxoplasma gondii The band at 29.9 kDa was consistent with the predicted relative molecular weight of TgAQP. Immunofluorescence showed that the protein recognized by the antisera was located in the cytoplasm of T. gondii. Conclusion The polyclonal antibodies against aquaporin of Toxoplasma gondii were prepared, which laid the foundation for further study on the biological functions and metabolism of aquaporins of Toxoplasma gondii.