【摘要】 为了解牛源犬新孢子虫AMA1基因蛋白特性及免疫原性,本试验以重组质粒PVAX1-NcAMA1为模板,PCR扩增NcAMA1基因,亚克隆至pGEX-4T-1表达载体;表达、纯化NcAMA1重组蛋白,并应用弗氏佐剂制备NcAMA1重组蛋白亚单位疫苗,接种BALB/c小鼠,间接ELISA方法检测小鼠血清抗体水平,用ELISA方法检测IFN-γ、IL-4表达水平。结果显示,表达的NcAMA1重组蛋白相对分子质量约为94 000(GST约为26 000、NcAMA1约为68 000),NcAMA1重组蛋白亚单位疫苗接种BALB/c小鼠后,能够诱导BALB/c小鼠产生较高的体液免疫水平和细胞免疫水平。本研究为利用该重组蛋白建立免疫学诊断方法及制备抗犬新孢子虫新型亚单位疫苗奠定了基础。更多还原

【Abstract】 In order to understand the characteristics of bovine Neospora caninum AMA1 protein and make a analysis of its immunogenicity,PVAX1-NcAMA1 recombinant plasmid was taken as a template,NcAMA1 gene was amplified and subcloned into PGEX-4T-1expression vector.Expressed and purificated recombinant NcAMA1 protein,preparated NcAMA1 recombinant protein subunit vaccine mixed Freund’s adjuvant to immune BALB/c mice,then used indirect ELISA method to analyze serum antibody levels.IFN-γand IL-4expression levels were analyzed by ELISA method.The results showed that the relative molecular mass of recombinant protein NcAMA1 was about 94 000(GST about 26 000,NcAMA1 about 68 000).NcAMA1 recombinant protein subunit vaccine were able to improve the humoral and cellular immunity level.This study laid the foundation for establishing immunological diagnostic methods and preparation of novel subunit vaccine anti-neospora by the recombinant protein.更多还原