柔嫩艾美耳球虫SAG2基因的克隆及其在卡介苗中表达  

Cloning of Eimeria tenella SAG2 Gene and Its Expression in BCG

作　　者：曹利利[1] 姚新华[1] 郭衍冰[1] 王英贺 任科研[1,3] 魏峰[2] 苑淑贤[1] 

机构地区：[1]吉林省畜牧兽医科学研究院,吉林长春130062 [2]吉林农业大学生命科学学院,吉林长春130118 [3]吉林省畜牧兽医总站,吉林长春130062

出　　处：《动物医学进展》2015年第9期13-17,共5页Progress In Veterinary Medicine

基　　金：吉林省科技发展计划项目(201205071);吉林省科技攻关计划项目(20150204070NY)

摘　　要：为获得柔嫩艾美耳球虫SAG2的克隆株并在卡介苗中表达该蛋白,根据柔嫩艾美耳球虫SAG2基因序列和表达载体图谱设计特异性引物,引入HindⅢ和EcoRⅠ酶切位点,RT-PCR扩增SAG2基因,连接到pMD18-T载体。双酶切后,将SAG2基因插入到大肠埃希菌-分支杆菌整合表达载体pMV361中,获得重组质粒pMV361-SAG2,经电穿孔转染到卡介苗中,表达产物经SDS-PAGE和Western blot分析。结果成功构建了重组质粒pMD18-T-SAG2和pMV361-SAG2,SAG2在卡介苗中高效表达,表达产物能够被柔嫩艾美耳球虫阳性血清识别,表明具有良好的免疫原性,为下一步重组卡介苗对鸡球虫病的免疫保护研究奠定了基础。The purpose of this study was to obtain the clone of Eimeria tenella SAG2 gene and express it in BCG. Specific primers were designed according to the sequences of E. tenella SAG2 gene and the expres- sion vector,the restriction enzyme sites of HindⅢ and EcoR I were inserted into primers. SAG2 gene was amplified by RT-PCR, and then ligated into pMD18-T vector. The SAG2 fragment was obtained after digesting by double restriction enzymes,and then SAG2 gene was inserted into Escherichia coli-Mycobacteri um integration pMV361 expression vector to obtain the recombinant plasmid pMV361-SAG2. The recombi- nant plasmid pMV361-SAG2 was transfected into BCG by electroporation, the recombinant protein was analysis by SDS-PAGE and Western blot. As results, the plasmids pMD18-T-SAG2 and pMV361-SAG2 were constructed successfully, the recombinant protein SAG2 was highly expressed in BCG,and it can be recognized by Eirneria tenella positive serum. The results suggested that Eimeria tenella SAG2 could be expressed in BCG, and there is a good immunogenicity. This study will make a good foundation to study the protective effect of recombinant BCG vaccine against coccidiosis in chickens in the future.

关 键 词：柔嫩艾美耳球虫 SAG2基因 卡介苗 表达 

分 类 号：S852.723[农业科学—基础兽医学]