重组恶性疟原虫醛缩酶鉴定及其单克隆抗体的制备  

Identification of Recombinant Aldolase of Plasmodium falciparum and its Monoclonal Antibody Preparation

作　　者：张瑞娟[1] 朱淮民[1] 郑徽[1] 宁北芳[1] 

机构地区：[1]第二军医大学病原生物学教研室

出　　处：《中国寄生虫学与寄生虫病杂志》2006年第3期196-199,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家"十五"科技攻关项目(2001BA705B09)~~

摘　　要：目的鉴定重组表达的恶性疟原虫醛缩酶(ALD),制备针对此酶的单克隆抗体。方法用PCR法扩增恶性疟原虫海南株ALD基因,经大肠埃希菌表达并纯化的ALD免疫BALB/c小鼠,腹腔注射免疫3次,每次间隔2周,加强免疫后3d取免疫小鼠脾细胞制备单克隆抗体。同时用获得的免疫血清进行间接荧光抗体试验(IFAT)和蛋白质印迹(Westerntblotting)分析。结果ELISA检测表明,小鼠能产生较高的针对ALD免疫应答,3次免疫后血清中特异性抗体滴度达1∶105,IFAT显示免疫血清能特异性识别疟原虫体内的抗原;Westernblotting分析显示免疫血清识别的疟原虫蛋白相对分子质量(Mr)约41000;所制备的免疫血清与人红细胞内醛缩酶无交叉反应。经ELISA检测3次,筛选获得7株分泌针对ALD的单克隆抗体的杂交瘤细胞株,其中3株分泌的单克隆抗体能识别培养的恶性疟原虫;抗体亚型鉴定结果显示均为IgG1型。结论本实验构建并表达了重组疟原虫糖酵解醛缩酶,并获得特异性的单克隆抗体。Objective To identify the recombinant aldolase （ALD） of Plasmodium falciparum, and to develop monoclonal antibodies （McAbs） against the recombinant ALD. Methods ALD gene was amplified by PCR from genomic DNA of FCCI/HN strain, and expressed in E.coli DH5α. BALB/c mice were immunized with the recombinant ALD of P. falciparum via celiac injection for 3 times with 2 weeks interval. Three days after a booster injection, spleen ceils of the immunized mice were used for producing McAbs. The immune serum was tested by IFAT and Western blotting. Results BALB/c mice immunized with purified aldolase protein developed strong immune response to the antigen, and the titer of specific antibody reached 1：105 in all immune sera after the third immunization. Moreover, immune sera specifically recognized the cultured P. falciparum. Western blotting showed that the immune sera recognized specifically a Mr 41 000 band of crude malaria antigen. No cross-reaction with human red cells was detected. Seven positive hybridoma cell lines were obtained after 3 rows of selection. All the McAbs＇ subclasses belong to IgGl. IFAT showed that only 4 McAbs could recognize the cultured P.falciparum. Conclusion Plasmodial aldolase has been successfully expressed and purified, and the established hybridoma cell lines can secrete McAbs specific to the aldolase of P. falciparum.

关 键 词：恶性疟原虫 醛缩酶 基因表达 单克隆抗体 

分 类 号：R382.312[医药卫生—医学寄生虫学] R392.11[医药卫生—基础医学]