猪弓形虫重组MIC3蛋白单克隆抗体的制备及其初步应用  

Preparation and Preliminary Application of Monoclonal Antibodies against rMIC3 of Toxoplasma gondii

作　　者：白昀[1,2] 王海燕[1] 王占伟[1,2] 纪燕[3] 吴叙苏[1] 刘冬霞[1] 邵国青[1] 

机构地区：[1]江苏省农业科学院兽医研究所.农业部兽用生物制品工程技术重点实验室.国家兽用生物制品工程技术研究中心,南京210014 [2]南京天邦生物科技有限公司,南京211102 [3]南京农业大学,南京210095

出　　处：《畜牧兽医学报》2013年第10期1622-1628,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：江苏省科技支撑计划(社会发展)项目(BE2010758)

摘　　要：为获得针对猪弓形虫MIC3蛋白的单克隆抗体,为进一步研制免疫金标试纸条提供材料,作者以纯化的重组MIC3蛋白免疫BALB/c小鼠,运用杂交瘤技术制备单克隆抗体(McAb)。经ELISA筛选阳性克隆和亚克隆建株,体内诱生腹水法制备McAb,测定其亚类、效价,进行Western blot分析,并利用胶体金颗粒标记的McAb制备免疫金标试纸条。成功制备了2株稳定分泌抗MIC3蛋白单克隆抗体的杂交瘤细胞株B7和D3,2株McAb的免疫球蛋白亚类均为IgG2a,其ELISA腹水效价分别为1∶64万和1∶192万,Western blot结果表明,2株McAbs腹水均能分别与纯化的重组MIC3蛋白和猪弓形虫全虫蛋白中大小为56和38ku左右的蛋白发生特异性反应;用纯化后McAb标记的胶体金颗粒制备的免疫金标试纸条可以检测到最低浓度为4×104 CFU·mL-1的弓形虫速殖子。本研究为猪弓形虫病的现场诊断提供了有效的方法,显示出很好的临床应用前景。MIC3 has high immunoreactivity, and plays an important role in recognition, adhesion and invasion for Toxoplasrna gondii to host cells. To prepare the monoclonal antibody （McAbs） against recombinant MIC3 （rMIC3） of T. gondii, we expressed rMIC3 in Escherichia coli, and purified rMIC3 was immunized to BALB/c mice. B lymphocytes hybridization technique was ap- plied to prepare the aimed McAb. Positive clones were screened with ELISA, and then were sub- cloned to establish stable cell lines. Ascites were induced to produce the McAbs, and its specifici- ty was identified by Western blot analysis. The gold immunochromatographic lateral flow assay system was developed. The results indicated that two stable hybridoma cell lines （B7 and D3） were obtained. The McAbs subclasses all belong to IgG2a and their ELISA titers of the ascitefluid were 1.640 000 and 1.1920 000, respectively. Western blotting analysis confirmed that the two McAbs all can identify the protein MIC3 from lysed taehyzoites and purified rMIC3. Purified polyclone antibody D3 was labeled with colloidal gold particle, and the minimum detectable limit of tachyzoite was 4 × 10^4 CFU . mL -1. This study laid the foundation for further study on related applications.

关 键 词：岗地弓形虫 MIC3蛋白 单克隆抗体 胶体金 

分 类 号：S852.7[农业科学—基础兽医学]