靶向DEC-205的弓形虫SAG1 DNA疫苗的构建及鉴定  

Construction and identification of a Toxoplasma gondii SAG1 DNA vaccine targeting DEC-205

作　　者：林霞琳 黄春芳 陈育琪 李丽娜 魏坤亚 陈欢 黄志坚 殷光文 LIN Xialin;HUANG Chunfang;CHEN Yuqi;LI Lina;WEI Kunya;CHEN Huan;HUANG Zhijian;YIN Guangwen(College of Animal Science,Fujian Agriculture and Forestry University,Fujian Animal Medicine Engineering Laboratory,Fuzhou 350002,China)

机构地区：[1]福建农林大学动物科学学院福建省动物药物工程实验室,福州350002

出　　处：《黑龙江畜牧兽医》2020年第20期72-75,80,159,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金面上项目(31872466);福建省科技厅引导性项目(2019N0005);福建省家禽产业技术体系建设项目(2019-2022);福建农林大学科技创新专项(CXZX2019070G)。

摘　　要：为了构建新型弓形虫DNA疫苗并鉴定,试验以弓形虫SAG1作为疫苗候选抗原,融合小鼠树突状细胞(DC)表面分子DEC-205的单链抗体(single chain antibody fragment against DEC-205,scFv DEC-205),构建可表达弓形虫速殖子的表面抗原SAG1和scFvDEC-205-SAG1的DNA质粒,对该质粒进行酶切鉴定,同时用该质粒对真核细胞进行转染,对目标蛋白的表达情况进行鉴定。结果表明:经电泳、双酶切及测序证实目的基因大小与实际相符,重组质粒pVAX1-scFvDEC205-SAG1构建成功;经Western-blot和细胞免疫荧光试验证实重组质粒pVAX1-scFvDEC205-SAG1可成功表达目的蛋白。说明成功构建重组DNA疫苗pVAX1-scFvDEC205-SAG1并在体外表达。The objective of this study was to construct and identify a novel DNA vaccine of Toxoplasma gondii(T.gondii),SAG1 was selected as a vaccine candidate antigen and the single-chain antibody fragment(scFv)against DEC-205,a mouse dendritic cell(DC)surface molecule was fused to construct a DNA plasmid expressing T.gondii surface antigens SAG1 and scFvDEC-205-SAG1.The plasmid was identified by enzyme digestion and transfected into eukaryotic cells to identify the expression of the target protein.The results showed that the size of the target gene was consistent with the reality by electrophoresis,double-enzyme digestion and sequencing,and the recombinant plasmid pVAX1-scFvDEC205-SAG1 was successfully constructed.Western-blot and immunofluorescence assay confirmed that the recombinant plasmid pVAX1-scFvDEC205-SAG1 could successfully express the target protein.It indicated that the recombinant plasmid pVAX1-scFvDEC205-SAG1 was successfully constructed and expressed in vitro.

关 键 词：弓形虫 DNA疫苗 SAG1抗原 树突状细胞 DEC-205单链抗体 重组质粒 

分 类 号：S852.72[农业科学—基础兽医学] Q78[农业科学—兽医学]