弓形虫TgMIC16多克隆抗体制备 纯化及其在亚细胞定位中的应用

Preparation and purification of polyclonal antibody against Toxoplasma gondii microneme protein 16 and its application in subcellular localization

目的制备并纯化弓形虫微线蛋白16(TgMIC16)兔源多克隆抗体,并利用制备的多克隆抗体对该蛋白进行亚细胞定位。方法利用原核表达、纯化的TgMIC16重组蛋白与等体积弗氏佐剂混合,背部皮下、多点注射免疫新西兰大白兔,于第3次加强免疫后的第14天收集兔血清,Protein A亲和纯化柱纯化,用ELISA和蛋白质印迹(Western blotting)分别检测抗体效价和抗体特异性。用弓形虫RH株速殖子感染人包皮成纤维(HFF)细胞,以制备的多克隆抗体为一抗,采用间接免疫荧光(IFA)检测TgMIC16在感染细胞中的定位。结果间接ELISA结果显示,制备的TgMIC16多克隆抗体滴度为1∶512 000;Western blotting结果显示,TgMIC16抗体特异性良好;IFA结果显示,TgMIC16定位在弓形虫微线体。结论本研究制备并纯化了抗弓形虫TgMIC16兔源多克隆抗体,并成功应用于TgMIC16在弓形虫微线体的免疫荧光定位。

Objective To prepare and purify the rabbit anti-TgMIC 16 polyclonal antibody, so as to apply it in subcellular localization. Methods New Zealand white rabbits were immunized with purified recombinant TgMIC16 mixing with the same volume of Freund＇ s adjuvant for three times, respectively. The rabbit serum was collected on the 14th day after the last immunization. The polyclonal antibody in rabbit serum was purified with Protein A affinity purification column. ELISA and Western blotting were used to detect the antibody titer and specificity of polyclonal antibody. The polyclonal antibody was used to the localization of TgMIC16 by the immunofluorescenee method. Results Indirect ELISA showed that the antibody titer was 1 ： 512 000. Western blotting showed that the recombinant TgMIC 16 protein was recognized by the specific polyclonal antibody. IFA showed that TgMIC16 was located in the microneme of Toxoplasma gondii. Conclusion The rabbit anti-TgMIC16 is prepared and purified, and successfully applied to immunofluorescence localization of TgMIC16 in T. gondii.