| 抗隐孢子虫鼠基因型CP15/60蛋白单克隆抗体的制备与鉴定 |
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| 引用本文： | 李正锋,陈兆国,何生虎,米荣升.抗隐孢子虫鼠基因型CP15/60蛋白单克隆抗体的制备与鉴定[J].动物医学进展,2010,31(10). |
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| 作者姓名： | 李正锋 陈兆国 何生虎 米荣升 |
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| 作者单位： | 1. 宁夏大学农学院,宁夏银川,750021
2. 中国农业科学院上海兽医研究所,农业部动物寄生虫学重点开放实验室,中国农业科学院动物源性食品安全研究中心,上海,200241 |
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| 基金项目： | 国家科技支撑计划项目，国家863项目，上海市科技兴农重点攻关项目(沪农科攻字 |
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| 摘 要： | 为获得高特异性的抗隐孢子虫单克隆抗体(monoclonal antibody,McAb),以原核表达的隐孢子虫鼠基因型(Cryptosporidium mouse genotype)子孢子表面抗原CP15/60为免疫原,免疫Balb/c小鼠。取免疫鼠脾细胞与SP2/0细胞进行融合,用间接ELISA方法进行抗体效价测定,有限稀释法进行亚克隆。以常规染色体分析法进行杂交瘤细胞的染色体分析,用McAb亚类鉴定试剂盒鉴定McAb亚型、间接ELISA法测定相对亲和力及交叉反应性、Western blot检测抗体的特异性。结果获得了2株能稳定分泌抗重组CP15/60(recombinant CP15/60,rCP15/60)的单克隆抗体杂交瘤细胞株,分别命名为B4F7和D8H5,其腹水抗体效价均达到1∶107;染色体数目B4F7株为97条±4条、D8H5株为90条±7条,且观察到标志染色体;McAb亚型均为IgG1,轻链均为κ链;相对亲和力常数B4F7株为0.009μg/mL、D8H5株为0.016μg/mL;2种McAb均与rCP15/60蛋白发生特异性反应,与柔嫩艾美耳球虫卵囊可溶性蛋白、弓形虫裂殖子蛋白以及日本血吸虫虫体蛋白均无交叉反应,证实本试验获得了2株高特异性的抗隐孢子虫McAb。
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| 关 键 词： | 隐孢子虫 CP15/60蛋白 单克隆抗体 |
Preparation and Identification of Monoclonal Antibodies Against CP15/60 of Cryptosporidium Mouse Genotype |
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| LI Zheng-feng,CHEN Zhao-guo,HE Sheng-hu,MI Rong-sheng.Preparation and Identification of Monoclonal Antibodies Against CP15/60 of Cryptosporidium Mouse Genotype[J].Progress In Veterinary Medicine,2010,31(10). |
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| Authors: | LI Zheng-feng CHEN Zhao-guo HE Sheng-hu MI Rong-sheng |
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| Institution: | LI Zheng-feng1,CHEN Zhao-guo2,HE Sheng-hu1,MI Rong-sheng2 (1.College of Agriculture,Ningxia University,Yinchuan,Ningxia,750021,China,2.Animal-borne Food Safety Research Center of Chinese Academy of Agricultural Sciences,Key Laboratory of Animal Parasitology of the Ministry of Agriculture,Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai,200241,China) |
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| Abstract: | In order to obtain specific monoclonal antibody(McAb)against Cryptosporidium,Balb/c mice were immunized with recombinant CP15/60(rCP15/60)protein of Cryptosporidium mouse genotype expressed in Escherichia coli BL21(DE3).The splenocytes of immunized mice were fused with SP2/0 cells.Anti-CP15/60 hybridoma cells were screened by indirect ELISA.Hybridoma cells were sub-cloned by limiting dilution assay.The chromosome of hybridoma cells were counted.McAb subtype was identified by isotype testing kit,affinity and... |
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| Keywords: | Cryptosporidium CP15/60 protein monoclonal antibody |
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