刚地弓形虫TgPRF的原核表达、多克隆抗体制备与应用  

Preparation and its application of polyclonal antibody against Toxoplasma gondii TgPRF after fusion expression in prokaryotic system

作　　者：闫爱霞 邹洋 黄敏君 李晶晶 李威 田小军 贾永根 谷俊朝 YAN Aixia;ZOU Yang;HUANG Minjun;LI Jingjing;LI Wei;TIAN Xiaojun;JIA Yonggen;GU Junchao(Beijing Friendship Hospital,Capital Medical University,Beijing Tropical Medicine Research Institute,Beijing Key laboratory for Prevention and Treatment of Tropical Disease,Beijing 100050,China)

机构地区：[1]首都医科大学附属北京友谊医院北京热带医学研究所热带病防治研究北京市重点实验室

出　　处：《中国热带医学》2019年第7期634-637,共4页China Tropical Medicine

基　　金：北京市自然科学基金资助项目(No.7182023);首都医科大学附属北京友谊医院科研启动基金资助项目(No.yyqdkt2017-3)

摘　　要：目的原核表达弓形虫TgPRF蛋白,制备多克隆抗体并评价其作为内参抗体的可行性。方法以刚地弓形虫RH株速殖子cDNA为模板,PCR扩增TgPRF编码基因,克隆至pGEX-4T-1载体,转化至大肠埃希氏菌(Escherichia coli)BL21。经IPTG诱导表达后,SDS-PAGE分析重组蛋白表达情况。利用GST标签亲和层析法纯化重组蛋白。以纯化后蛋白为抗原免疫新西兰大白兔,制备抗TgPRF多克隆抗体。收集弓形虫RH株速殖子,以制备的多克隆抗体为一抗,Western blotting检测抗体特异性及效价。收集ATc调控下不同时间点的弓形虫iKO-Ty-TgAPH虫株速殖子进行Western blotting,以抗Ty和抗TgPRF抗体为一抗,检测TgAPH和TgPRF表达量的变化。结果 SDS-PAGE显示:TgPRF在诱导4 h后表达量趋于饱和,相对分子量为52 ku,且呈可溶性表达。弓形虫RH株速殖子总蛋白进行的Western blotting显示:抗TgPRF多克隆抗体可识别内源性的TgPRF,且该多克隆抗体稀释至1∶10 000时仍可见明显条带;iKO-Ty-TgAPH虫株速殖子总蛋白进行的Western blotting显示:TgAPH的表达量随ATc作用时间的增加呈递减趋势,而TgPRF表达量基本维持恒定。结论本实验制备的抗TgPRF多克隆抗体特异性和效价良好,可作为衡量弓形虫特定蛋白表达量的内参抗体,也可直接检测内源性TgPRF的表达。Objective To generate rabbit polyclonal antibodies against the recombinant protein GST-TgPRF to be used as a loading control antibody. Methods Using the tachyzoite cDNA of Toxoplasma gondii RH strain as template, the TgPRF coding gene was amplified by PCR, cloned into pGEX-4T-1 vector, and transformed into Escherichia coli BL21. After IPTG induced expression, SDS-PAGE was used to analyze the expression of recombinant protein. The recombinant protein was purified by GST label affinity chromatography. Anti-TgPRF polyclonal antibody was prepared by immunizing New Zealand white rabbits with purified protein antigen. The tachyzoites of T. gondii RH strain were collected, and the polyclonal antibodies prepared were used as primary antibodies, and the specificity and titer of the antibodies were detected by Western blotting.The tachyzoites of Toxoplasma iKO-Ty-TgAPH strains at different time points regulated by ATc were collected for Western blotting, and Ty and anti-TgPRF were used as primary antibodies to detect the changes in the expression levels of TgAPH and TgPRF. Results SDS-PAGE showed a 52 ku band of expressed proteins post IPTG-induction 4 h. Western blotting of the total protein of tachyzoites of Toxoplasma RH strain showed that anti-TgPRF polyclonal antibody could recognize endogenous TgPRF, and obvious bands could be seen when the polyclonal antibody was diluted to 1∶10 000;Western blotting of the total protein of tachyzoites of iKO-Ty-TgAPH showed that the expression of TgAPH decreased with the increase of ATc, while the expression of TgPRF remained basically constant. Conclusion The rabbit anti-TgPRF polyclonal antibody generated in this experiment showed a good specificity and sensitivity. It can be used as an internal control in western blot analyses and may be used as well as in other immunological applications.

关 键 词：刚地弓形虫 弓形虫前纤维蛋白 多克隆抗体 内参抗体 

分 类 号：R382.5[医药卫生—医学寄生虫学]