细粒棘球绦虫水通道蛋白9的多肽特征及免疫定位研究

目的 研制细粒棘球绦虫水通道蛋白9 (EgAQP9)的特异性多肽抗体,并用于检测其在棘球蚴囊壁、生发细胞及原头蚴中的组织分布情况。 方法 根据EgAQP9特异氨基酸序列设计合成B细胞抗原多肽,再用合成的多肽偶联KLH作为免疫原注射免疫新西兰兔以制备多克隆抗体。酶联免疫吸附试验(ELISA)方法检测多肽抗体滴度,蛋白免疫印迹法(Western blot)测定多肽抗体免疫活性,免疫荧光实验分析EgAQP9的亚细胞定位,免疫组化实验分析EgAQP9组织定位。 结果 免疫新西兰兔成功制备出多肽抗体;间接ELISA检测其多肽抗体效价高达1∶256 000;Western blot结果显示多肽抗体能够特异识别细粒棘球绦虫中36 kDa处的条带,与EgAQP9预测的相对分子量大小相符;免疫荧光实验结果显示EgAQP9分布于棘球蚴生发细胞的胞质、胞膜中;免疫组化实验显示该蛋白主要分布在原头蚴表面及棘球蚴囊泡生发层。结论 本研究以制备的EgAQP9兔源多克隆抗体定位了AQP9在棘球蚴生发细胞、原头蚴及棘球蚴囊壁中的分布状况。

Peptide characterization and immunolocalization of Echinococcus granulosus aquaporin 9

A specific polypeptide antibody of Echinococcus granulosus aquaporin 9(EgAQP9)was developed and used to detect the tissue distribution of this protein in the germinal cells,protoscoleces and hydatid cyst walls of Echinococcus granulosus.The B cell antigen polypeptide was designed on the basis of the amino acid sequence of EgAQP9,and the polyclonal antibodies was prepared by using the synthesized polypeptide coupled with KLH as an immunogen to immunize New Zealand rabbits.The multi-antibody titer was determined with ELISA,the immunoactivity was analyzed with western blotting,the subcellular localization of EgAQP9 was determined with immunofluorescence assays,and the tissue localization of EgAQP9 was determined with immunohistochemistry assays.The polyclonal antibody was successfully prepared,with a titer as high as 256000,according to ELISA.Western blotting revealed that the prepared antibody allowed for specific identification of bands at 36 kDa in natural E.granulosus;this mass was similar to the predicted relative molecular weight of EgAQP9.Furthermore,immunofluorescence assays indicated that EgAQP9 was distributed in the cytoplasm and membranes of the germinal cells of hydatids,and immunohistochemistry assays showed that the protein was mainly distributed at the surfaces of the protoscoleces and in the germinal layers of the hydatid cysts.The prepared EgAQP9 rabbit-derived polyclonal antibody was used to determine the distribution of EgAQP9 protein in the germinal cells,protoscoleces and cyst walls of E.granulosus hydatids.