抗弓形虫水通道蛋白多肽抗体的制备及鉴定

Preparation and identification of anti-TgAQP peptide antibody

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。

In this study, we intended to prepare anti-Toxoplasma gondii aquaporin （TgAQP） peptide antibody which was used to the application in the detection of the aquaporin expression and its subeellular localization of Toxoplasma gondii （T. gondii） ME49 strain. The B cell peptide antigen was designed based on the TgAQP amino acids sequence. After the pep- tide antigen was conjugated to the KLH, the fusion antigen was injected into New Zealand rabbits to prepare polyclonal anti- body, followed by identification of ELISA, Western-blotting and immunofluorescence assays. The ELISA showed that the titer of antbTgAQP antibody was about 1 ~ 40 000. Western blotting revealed the specific affinity of the antigen to polyclonal anti- body at 29.9 kDa protein T. gondii. The protein detected by the indirect immunofluorescence assays was distributed in the cy- toplasm of the parasite. Thus farm the anti-TgAQP polyclonal antibody was successfully prepared, providing a useful tool for further study of biological function and metabolic characteristics of TgAQP.