학술논문
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 AMA1 Gene
Document Type
Academic Journal
Author
李航; 姜利建; 刘晋宇; 兰岚; 凌芳芳; 石甜甜; 张贺洋; 贾立军; LI Hang; JIANG Li-jian; LIU Jin-yu; LAN Lan; LING Fang-fang; SHI Tian-tian; ZHANG He-yang; JIA Li-jun?
Source
中国畜牧兽医. 43(12):3336-3342
Subject
Language
Chinese
ISSN
1671-7236
Abstract
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/TgA-MA1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.
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.