新孢子虫与弓形虫交叉抗原AMA1基因重组腺病毒的构建及免疫应答分析

Construction and Analysis of Immune Response of Neospora caninum and Toxoplasma gondii Cross Recombinant Adenovirus Antigen AMA1Gene

为构建新孢子虫和弓形虫AMA1基因重组腺病毒,并分析其免疫原性,本试验根据新孢子虫和弓形虫AMA1基因序列的开放阅读框,设计新孢子虫和弓形虫交叉抗原AMA1基因通用引物,构建重组克隆质粒pMD18T-NcAMA1、pMD18T-TgAMA1及重组腺病毒穿梭质粒ADV4-Nc/TgAMA1,将ADV4-Nc/TgAMA1和骨架质粒pacAd5线性化后共转染293T细胞,包装Ad5-Nc/TgAMA1重组腺病毒,测定病毒滴度后,收集病毒液接种BALB/c小鼠,间接ELISA检测小鼠血清IgG抗体水平。结果显示,Nc/TgAMA1在Ad5-Nc/TgAMA1重组腺病毒中获得表达,测定Ad5-Nc/TgAMA1重组腺病毒滴度为109 PFU/mL,接种BALB/c小鼠后,Ad5-Nc/TgAMA1接种组IgG抗体水平明显高于pVAX1-Nc/TgAMA1质粒组和PBS对照组。结果表明,构建的Ad5-Nc/TgAMA1重组腺病毒能诱导小鼠产生特异性体液免疫应答。本试验为新孢子虫和弓形虫交叉抗原AMA1基因重组腺病毒载体疫苗的研制奠定了基础。

In order to establish AMA1 recombinant adenovirus of Neospora caninum （N .cani-num）and Toxoplasma gondii （T .gondii）,and analyze the immunogenicity of it,cross universal primers were designed according to the open reading frame of N .caninum and T .gondii AMA 1 gene sequences.Based on pMD18T-NcAMA1 and pMD18T-TgAMA1 cloning plasmid,recombi-nant adenovirus shuttle plasmid ADV4-Nc/TgAMA1 was constructed.Then,ADV4-Nc/TgA-MA1 and pacAd5 backbone plasmid were linearized and co-transfected 293T cells.After packa-ging recombinant adenovirus and measuring the virus titer,collected virus was inoculated into BALB/c mice,confirmed the IgG antibody levels by indirect ELISA method.The results showed that Nc/TgAMA1 was expressed in Ad5-Nc/TgAMA1 recombinant adenovirus,Ad5-Nc/TgAMA1 re-combinant adenovirus titer was 109 PFU/mL.IgG antibody levels in the Ad5-Nc/TgAMA1 vaccinated group were significantly higher than pVAX 1-Nc/TgAMA1 plasmid group and PBS control group.This result indicated that the constructed Ad5-Nc/TgAMA1 recombinant adenovirus could induce specific hu-moral immune response in mice,this research laid a solid foundation for the development of a recombi-nant adenovirus vaccine against N .caninum and T.gondii.