重组鹅源新城疫病毒HN基因乳酸菌的构建  

Construction of recombinant Lactobacillus containing HN gene of Newcastle disease virus from goose

作　　者：胡静涛[1] 郭衍冰 杨文涛[1] 石少华[1] 王春凤[1] 丁壮[3] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]吉林省畜牧科学研究所,吉林长春130062 [3]吉林大学动物医学学院,吉林长春130062

出　　处：《中国兽医科学》2015年第12期1242-1246,共5页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31302061,313272541,313272552,31472195,31402195);吉林省科技支撑计划重点科技攻关项目(20150204025NY);教育部高校博士点基金博导类项目(20110061110005)

摘　　要：构建了重组鹅源新城疫病毒(NDV)HN基因乳酸杆菌后,将HN基因亚克隆至可在乳酸杆菌和大肠杆菌中穿梭表达的载体pSIP409中。此后,将构建的阳性重组体pSIP409-HN采用电转化方法转化至植物乳酸杆菌感受态细胞中。利用SDS-PAGE和Western-blot检测HN蛋白的表达情况。为明确HN蛋白的表达位置,利用流式细胞术检测了重组菌细胞是否有HN蛋白的表达。结果显示,克隆了NDV HN基因,构建了阳性重组质粒pSIP409-HN;鉴定结果表明,阳性重组质粒pSIP409-HN被成功电转化至乳酸杆菌感受态细胞中。SDS-PAGE和Western-blot分析结果表明,构建的重组菌可以表达与HN蛋白已知大小相符的条带(66ku),且该蛋白能与NDV阳性抗体反应,具有反应原性。流式细胞术检测结果表明,HN蛋白被表达于菌体细胞表面,可作为黏膜疫苗的候选抗原。To construct recombinant Lactobacillus containing a HN gene of Newcastle disease virus（NDV）,the HN gene was sub-cloned into Lactobacillus shuttle vector to construct a recombinant plasmid（named as pSIP409-HN）.And then the recombinant plasmid pSIP409-HN was elec-transformed into Lactobacillus plantarum competence cells.The electroporation result was testified by plasmid extraction and enzyme digestion analysis.SDS-PAGE and Western-blot were used to detect the expression of HN protein.In order to observe the expression of foreign gene fromLactobacillus anchored cell surface,the flow cytometry was used to detect the expression of HN protein on Lactobacillus strain cell surface.In result,the recombinant Lactobacillus containing NDV HN gene was constructed.Results of SDS-PAGE and Westernblot showed that recombinant Lactobacillus expressed a protein（nearly 66 ku in size）with the known HN protein and can react with NDV protein antibody.Importantly,HN protein could be expressed on the surface of Lactobacillus strain,which could be acted as the candidate of mucosal vaccine.

关 键 词：新城疫病毒 血凝素-神经氨酸酶基因 乳酸杆菌 表达 

分 类 号：S852.659.5[农业科学—基础兽医学]