抗微小隐孢子虫 Apple 结构域单克隆抗体的制备

摘要

采用PCR技术从已构建的微小隐孢子虫cDNA文库中扩增含Apple 结构域基因，构建pGEX-4T-1-Apple重组质粒，并进行原核表达及纯化，得到分子质量大小约为34 kDa的GST-Apple重组蛋白，以其为免疫原，免疫Balb/c小鼠，利用杂交瘤技术制备单克隆抗体，测定其免疫球蛋白亚类及效价，并用蛋白质印迹法（Western-blot）鉴定其特异性。结果共筛选出能稳定分泌抗重组Apple单克隆抗体的2株杂交瘤细胞株，抗体亚类均为IgG1， Western-blot分析显示2株单抗均能与重组蛋白GST-Apple发生特异性结合。为研究Apple结构域在隐孢子虫侵入过程中的功能提供有效工具。%Apple domain gene was amplified from cDNA library of Cryptosporidium parvum by PCR method. The recombinant plasmid pGEX-4T-1-Apple were constructed and then expressed in E. coli. The molecular weight of the recombination protein is 34 kDa.BALB/c mice were immunized with purified recombinant protein to prepare McAbs .The subtype and specificity of McAb were identified by kit and Western-blot respectively.Two hybridoma cell lines secreting monoclonal antibodies aganist Apple domain were obtained.The subtype of both McAbs are IgG1.Western-blot showed that the two McAbs reacted strongly and highly specific against GST-Apple recombination proteins.It would provide effective material to study the function of Apple domain during parasite attachment and host cell invasion.