兔抗弓形虫Ⅰ型液泡型质子焦磷酸酶多克隆抗体的制备及鉴定

摘要

Objective To prepare and characterize rabbit polyclonal antibodies against Toxoplasma gondii vacuolar proton pyrophosphatase type I (TgVP1). Methods and Results Two synthesized peptides TgVP1-1 and TgVP1-2 as the haptens were conjugated with KLH to immunize rabbits. Indirect ELISA showed that the titers of rabbit anti-TgVP1-1 polyclonal antibody and rabbit anti-TgVP1-2 polyclonal antibody reached 1:128 000. Western blotting results revealed that both purified polyclonal antibodies could specifically bind to a purified 85 kD T. gondii protein predicted as TgVP1. The protein detected by these two polyclonal antibodies was distributed in the cytoplasm of T. gondii tachyzoite, and this distribution pattern was consistent with that of acidocalcisome. Conclusion The peptide-based method of antibody generation is efficient and the obtained TgVP1 polyclonal antibodies possess a high specificity to facilitate further study of T. gondii acidocalcisome and the diagnosis of toxoplasmosis.%目的 制备兔抗弓形虫I型液泡型质子焦磷酸酶(TgVP1)多克隆抗体并鉴定其应用.方法 选择弓形虫ME49株TgVP1氨基酸序列中的两条多肽,TgVP1-1与TgVP1-2,进行人工合成,并将其与KLH偶联作为抗原免疫新西兰大耳白兔,收集血清制备多克隆抗体,并通过ELISA、Western blot、免疫荧光等方法进行鉴定.结果 经间接ELISA测定,兔抗TgVP1-1及TgVP1-2的多克隆抗体效价均达1:128 000;Western blot结果显示两种多抗均能识别弓形虫天然蛋白中85 000左右条带,与TgVP1预测相对分子质量大小相符,且特异性较好;通过免疫荧光技术检测到两株多抗识别的蛋白均定位于细胞质中,与酸性钙体的分布相同.结论 采用人工合成多肽为免疫原所获得的兔抗TgVP1多克隆抗体效价高,特异性好,弓形虫TgVP1和酸性钙体的深入研究奠定了基础,同时也为弓形虫病的诊断提供了新的有效工具.