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

doi:10.16431/j.cnki.1671-7236.2016.12.037

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (12): 3336-3342.doi: 10.16431/j.cnki.1671-7236.2016.12.037

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

李航, 姜利建, 刘晋宇, 兰岚, 凌芳芳, 石甜甜, 张贺洋, 贾立军

延边大学农学院动物医学系, 延吉 133002

收稿日期:2016-05-20 出版日期:2016-12-20 发布日期:2016-12-22
通讯作者: 贾立军 E-mail:lijunjia1015@sohu.com
作者简介:李航(1993-),女,吉林农安人,学士,研究方向:分子免疫学,E-mail:627105428@qq.com
基金资助:
国家自然科学基金（31160501、31360605）；吉林省重点科技攻关项目（20140204078NY）；吉林省教育厅重点项目（吉教科合字[2015]第1号）；吉林省自然科学基金（20160101201JC）

Construction and Analysis of Immune Response of Neospora caninum and Toxoplasma gondii Cross Recombinant Adenovirus Antigen AMA1 Gene

LI Hang, JIANG Li-jian, LIU Jin-yu, LAN Lan, LING Fang-fang, SHI Tian-tian, ZHANG He-yang, JIA Li-jun

Department of Veterinary Medicine, Agricultural College of Yanbian University, Yanji 133002, China

Received:2016-05-20 Online:2016-12-20 Published:2016-12-22

摘要/Abstract

摘要：

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10⁹PFU/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。

关键词: 新孢子虫; 弓形虫; AMA1基因; 重组腺病毒; 免疫应答

Abstract:

In order to establish AMA1 recombinant adenovirus of Neospora caninum (N.caninum) 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 AMA1 gene sequences. Based on pMD18T-NcAMA1 and pMD18T-TgAMA1 cloning plasmid, recombinant adenovirus shuttle plasmid ADV4-Nc/TgAMA1 was constructed. Then, ADV4-Nc/TgAMA1 and pacAd5 backbone plasmid were linearized and co-transfected 293T cells. After packaging 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 recombinant adenovirus titer was 10⁹ PFU/mL. IgG antibody levels in the Ad5-Nc/TgAMA1 vaccinated group were significantly higher than pVAX1-Nc/TgAMA1 plasmid group and PBS control group. This result indicated that the constructed Ad5-Nc/TgAMA1 recombinant adenovirus could induce specific humoral immune response in mice, this research laid a solid foundation for the development of a recombinant adenovirus vaccine against N. caninum and T. gondii.

Key words: Neospora caninum; Toxoplasma gondii; AMA1 gene; recombinant adenovirus; immune response

中图分类号:

S852.7

李航, 姜利建, 刘晋宇, 兰岚, 凌芳芳, 石甜甜, 张贺洋, 贾立军. 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[J]. 《中国畜牧兽医》, 2016, 43(12): 3336-3342.

LI Hang, JIANG Li-jian, LIU Jin-yu, LAN Lan, LING Fang-fang, SHI Tian-tian, ZHANG He-yang, JIA Li-jun. Construction and Analysis of Immune Response of Neospora caninum and Toxoplasma gondii Cross Recombinant Adenovirus Antigen AMA1 Gene[J]. , 2016, 43(12): 3336-3342.

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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

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