新孢子虫NcGRA2蛋白多克隆抗体的制备及其亚细胞定位

Polyclonal antibody preparation and subcellular localization of the NcGRA2 protein of Neospora caninum

目的制备重组新孢子虫NcGRA2t全蛋白去掉信号肽多克隆抗体并纯化,研究其在新孢子虫虫体内的定位。方法用新孢子虫NcGRA2t蛋白进行体外原核表达,纯化后免疫健康BALB/c小鼠,制备抗血清,采用Western blot分析抗体特异性,采用免疫荧光法检测NcGRA2蛋白在新孢子虫处于细胞内阶段和细胞外阶段时的亚细胞定位。结果Western blot检测,制备的鼠抗NcGRA2t多克隆抗体能与新孢子虫裂解抗原发生特异性反应。间接免疫荧光分析,NcGRA2蛋白在细胞内阶段和细胞外阶段的虫体细胞质中均呈现绿色荧光。结论本研究制备并纯化了新孢子虫NcGRA2蛋白多克隆抗体,用该抗体可检测到新孢子虫NcGRA2蛋白存在虫体的细胞质中。

Objectives To prepare a polyclonal antibody against Neospora caninum NcGRA2t(truncated NcGRA2 lacking a signal peptide)and to study the subcellular localization of the NcGRA2 protein of N.caninum. Methods The NcGRA2t-GST protein was expressed in vitro and purified using Glutathione Sepharose 4B.BALB/c mice were immunized intraperitoneally with the purified recombinant NcGRA2t-GST protein to prepare the polyclonal antibody.The subcellular localization of NcGRA2 in N.caninum was identified with indirect immunofluorescence using the prepared polyclonal antibody. Results The protein NcGRA2t-GST was obtained and purified at a temperature of 37℃.The NcGRA2t-GST protein band was detected at 46×103.Western blotting indicated that the mouse polyclonal antibody against the NcGRA2 tprotein reacted with the lysis antigen of N.caninum.Indirect immunofluorescence analysis indicated that the NcGRA2 protein displayed green fluorescence in the cytoplasm of both the intracellular stage and the extracellular stage of N.caninum. Conclusion This study prepared a polyclonal antibody against NcGRA2 t,and results suggested that the NcGRA2 protein is localized in the cytoplasm of N.caninum.