Abstract: Objective　To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of Toxoplasma gondii, and explore its preliminary applications. Methods　The GRA24 coding sequences of different T. gondii strains were aligned using the MEGA⁃X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET⁃28a vector, and the generated GRA24 truncated protein was transformed into Escherichia coli BL21. After induction by isopropyl⁃beta⁃D⁃thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate ⁃ polyacrylamide gel electrophoresis (SDS⁃PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA). Results　SDS⁃PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high⁃purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1∶208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different T. gondii strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following T. gondii invasion in host cells. Conclusions　The polyclonal antibody against the T. gondii GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.

Key words: Toxoplasma gondii, Dense granule protein 24, Polyclonal antibody, Immunofluorescence?assay

摘要： 目的　制备并鉴定鼠抗刚地弓形虫致密颗粒蛋白24（dense granule protein 24，GRA24）多克隆抗体，并探索其初步应用。方法　利用MEGA⁃X软件比对弓形虫不同虫株GRA24编码区序列，使用Protean软件分析GRA24蛋白优势肽段，通过PCR反应扩增编码该肽段的碱基序列，并连接至pET⁃28a载体中。将获得的GRA24截短蛋白原核表达质粒转化于大肠埃希菌BL21感受态细胞中，异丙基⁃β⁃D⁃硫代半乳糖苷（isopropyl⁃beta⁃D⁃thiogalactopyranoside，IPTG）诱导后采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate⁃polyacrylamide gel electrophoresis，SDS⁃PAGE）检测蛋白表达与纯化。使用纯化的GRA24截短蛋白皮下注射免疫BALB/c小鼠获得GRA24截短蛋白多克隆抗体，采用酶联免疫吸附试验（enzyme⁃linked immunosorbent assay，ELISA）检测抗体效价，采用Western blotting检测抗体特异性，并将该抗体应用于免疫荧光试验（immunofluorescence assay，IFA）。结果　SDS⁃PAGE结果表明成功构建重组质粒，考马斯亮蓝染色结果显示获得高纯度重组GRA24截短蛋白。ELISA结果显示，GRA24截短蛋白多克隆抗体效价在1∶208 400以上；Western blotting检测发现，该抗体可识别弓形虫不同虫株内源性GRA24蛋白，特异性识别重组GRA24截短蛋白；间接IFA检测发现，弓形虫入侵宿主细胞16 h后分泌的GRA24蛋白定位于宿主细胞核中。结论　成功制备广适性、高效价、强特异性的抗弓形虫GRA24多克隆抗体，可应用于Western blotting与IFA，为进一步研究GRA24功能奠定了基础。 　

关键词: 刚地弓形虫, 致密颗粒蛋白24, 多克隆抗体, 免疫荧光