一种柔嫩艾美耳球虫棒状体蛋白单抗的制备及初步应用

PREPARATION AND PRELIMIMARY APPLICATION OF MONOCLONAL ANTIBODY AGAINST A RHOPTRY PROTEIN OF EIMERIA TENELLA

利用原核表达的柔嫩艾美耳球虫棒状体蛋白(EtRP)的重组蛋白免疫BALB/c小鼠,经4次免疫后取脾脏制备免疫脾细胞,与小鼠骨髓瘤细胞SP2/0融合,经间接ELISA和Western blot筛选获得一株抗EtRP蛋白的特异性单克隆抗体细胞株,命名为2E3,亚类鉴定为IgG2a。细胞上清和小鼠腹水的效价分别为1∶40和1∶2.56×104。利用该单抗对EtRP在柔嫩艾美耳球虫子孢子中的分布进行分子定位,结果显示该蛋白主要分布于子孢子的顶端;而当子孢子在DMEM培养基中41℃孵育1 h后,该蛋白则分布于整个虫体。研究结果表明,成功制备了EtRP单克隆抗体,为进一步研究球虫相关蛋白功能奠定了基础。

BALB/c mice were immunized four times with E. coli expressed fusion protein to prepare specific monoclonal antibody （MAb） against a rhoptry protein of Eimeria tenella（EtRP） and to study distribution of the protein on the sporozoites. Cell fusion was conducted according to standard procedure. After screened by indirect ELISA and Western blot, one hybridoma clone that secreted specific MAb against EtRP was obtained. This MAb designated as 2E3 was characterized as IgG2a. The ELISA titers of culture supernatant and murine ascites were 1：40 and 1：2.56 × 10^4 , respectively. The distribution of EtRP on the sporozoites was investigated using the MAb 2E3. The results indicated that the protein was located at apical tips of sporozoites but became distributed in whole body of sporozoites when sporozoites were incubated in DMEM at 41 ℃ for 1 h.