ADV VP2主要抗原表位基因核酸疫苗与IL-12、IFN-γ细胞因子佐剂质粒的构建及鉴定  

Construction and Identification of Aleutian Disease Virus VP2 MajorAntigen Epitope Gene Nucleic Acid vaccine and IL-12,IFN-γ Cytokine Adjuvant Plasmids

作　　者：柏玲 朱言柱[1,2] 胡博 朱翔宇[1,2] 张海玲 廉士珍 张蕾[1,2] BAI Ling;ZHU Yan-zhu;HU Bo;ZHU Xiang-yu;ZHANG Hai-ling;LIAN Shi-zhen;ZHANG Lei(Institute of Special Wild Economic Animals and Plants,Chinese Academy of Agricultural Sciences,Changchun 130112,China;Key Laboratory of Special Animal Epidemic Disease,Ministry of Agriculture and Rural Affairs,Changchun 130112,China)

机构地区：[1]中国农业科学院特产研究所,吉林长春130112 [2]农业农村部经济动物疫病重点实验室,吉林长春130112

出　　处：《特产研究》2020年第2期1-8,14,共9页Special Wild Economic Animal and Plant Research

基　　金：中国农业科学院特产研究所科技创新工程协同创新项目(2018XTCX001)。

摘　　要：为研制安全有效的水貂阿留申病毒(ADV)VP2核酸疫苗,本研究构建了不含有形成抗原抗体复合物表位的VP2核酸疫苗以及IL-12、IFN-γ细胞因子真核表达质粒,在体外细胞水平鉴定上述目的基因的表达情况。首先,以各克隆质粒为模板,PCR扩增获得ADV的VP2主要抗原表位M、鼠IL-12和IFN-γ的基因片段,连接至pVAX1载体,经酶切及测序鉴定后,确定构建正确;其次,将真核表达质粒转染入293T细胞,通过间接免疫荧光及Western Blot分析表达情况。结果显示:pVAX1-M可正常表达,并且目的蛋白可与ADV阳性血清结合,具有良好的反应原性。含细胞因子的真核表达质粒pVAX1-IL及pVAX1-IF转染293T细胞后,通过间接免疫荧光、Western Blot及ELISA分析证明,目的蛋白可在体外正常表达,且具有生物学活性。本研究可为水貂阿留申病核酸疫苗深入研究奠定基础。In order to develop a safe and effective Aleutian disease virus(ADV)VP2 nucleic acid vaccine,we constructed a VP2 nucleic acid vaccine that did not contain antigen-antibody complex epitope and eukaryotic expression plasmids of IL-12 and IFN-γcytokines,and identified the expression of the above-mentioned target genes in vitro.First,each cloned plasmid was used as a template,and the gene fragments of ADV’s main VP2 epitope M,murine IL-12 and IFN-γwere obtained by PCR amplification.The fragments were ligated to the pVAX1 vector.The digestion and sequencing confirmed that the plasmids were successfully constructed.Second,the eukaryotic expression plasmids were transfected into 293 T cells,and the expression was analyzed by indirect immunofluorescence and Western Blot.The results showed that p VAX1-M can be expressed normally,and the target protein can bind to ADV-positive serum,and has good reactogenicity.After transfected 293 T cells with eukaryotic expression plasmids pVAX1-IL and p VAX1-IF containing cytokines,analysis of indirect immunofluorescence,Western Blot and ELISA proved that the target protein can be normally expressed in vitro and has biological activity.This study paved the way for the study of Aleutian disease nucleic acid vaccines.

关 键 词：水貂阿留申病毒 VP2 抗原抗体复合物 IL-12 IFN-Γ 核酸疫苗 

分 类 号：S852.65[农业科学—基础兽医学] Q786[农业科学—兽医学]