摘要
目的:制备并鉴定兔抗弓形虫CorA家族Mg^(2+)转运蛋白(TgMg)的特异性多克隆抗体。方法:设计并合成一段的具有较好免疫原性的、来源于TgMg氨基酸序列的短肽,短肽与KLH偶联后免疫新西兰大耳白兔,收集兔血清并分离纯化TgMg多克隆抗体,利用间接ELISA、Western印迹、间接免疫荧光等多种分子生物学方法鉴定多抗的效价、特异性及TgMg蛋白的亚细胞定位。结果:间接ELISA测定多抗效价为1∶160000;Western印迹显示该多抗能识别相对分子质量约为174×103的单一条带,表明抗体的特异性较好;用间接免疫荧光法发现TgMg定位于细胞质,与预期结果相符。结论:运用人工合成多肽结合经典的多克隆抗体制备技术制备了抗TgMg多克隆抗体,为深入研究TgMg蛋白的生物学特性及代谢功能提供了有效工具。
Objective： To prepare and identify rabbit polypeptide antibody（p Ab） against Cor A family Mg^（2＋） trans-porter protein in Toxoplasma gondii（Tg Mg）. Methods： We designed and synthesized a polypeptide from the aminoacid sequence of Tg Mg with good immunogenicity. The polypeptide was conjugated with keyhole limpet hemoyanin（KLH） as antigen for immunity to the New Zealand white rabbits. After purification of p Ab from the serum of rab-bit, we identified the titer and the specificity of this p Ab, as well as the intracellular location of Tg Mg by indi-rect ELISA, Western blot and indirect immunofluorescence assays respectively. Results： Indirect ELISA revealedthat the titer of anti-Tg Mg p Ab was 1∶160 000. Western blot assay showed that a prominent band of about 174 k Da, corresponding in size to that of Tg Mg, indicating the specificity of this p Ab. Immunofluorescence assayshowed that Tg Mg was localized to the cytoplasm as expected. Conclusion： We have prepared the anti-Tg Mg p Abusing synthesized peptide and classical p Ab preparation method, providing powerful tool for further exploring the bi-ological and metabolic functions of Tg Mg.
出处
《生物技术通讯》
CAS
2016年第6期778-782,共5页
Letters in Biotechnology
基金
国家自然科学基金(81171608)
广东省部产学研结合项目(2012B091100158)
关键词
弓形虫
Mg^(2+)转运蛋白
多克隆抗体
免疫荧光
Toxoplasma gondii
Mg^（2＋） transporter protein
polyclonal antibody
immunofluorescence
