摘要
目的分别构建细粒棘球绦虫Eg95重组非分泌型和分泌型卡介苗及耻垢分枝杆菌疫苗。方法分别以卡介苗(BCG)基因组DNA和PGEX-4T-Eg95为模板,PCR扩增获120bp的BCG抗原85B信号肽序列和423bp的Eg95基因序列。先将Eg95基因序列定向克隆至大肠埃希菌-分枝杆菌穿梭质粒pMV261,构建非分泌型重组质粒pMEg95。再将BCG-Ag85B信号肽序列定向克隆至pMEg95,构建分泌型重组质粒pSMEg95。电穿孔法将两型重组质粒分别导入BCG菌及耻垢分枝杆菌。结果双酶切、PCR扩增及测序鉴定证实,克隆基因Eg95序列和Ag85B信号肽序列正确插入载体PMV261,细粒棘球绦虫Eg95重组非分泌型和分泌型分枝杆菌疫苗构建成功。结论构建了含有Eg95基因序列和BCG-Ag85B信号肽序列的细粒棘球绦虫Eg95重组非分泌型和分泌型分枝杆菌疫苗。
To construct the recombinant secreted and non-secreted mycobacterium-Eg95 vaccines for Echinococcua granolosus ,the 120 bps of BCG-Ag85 signal sequence and the 423 bps of Eg95 gene sequence were amplified by PCR. Firstly, Eg95 gene fragment was cloned into E. coli-BCG shuttle vector pMV261 to obtain the recombinant non-secreted plasmid pMEg95. Next,the BCG-Ag85B signal sequence was cloned into plasmid pMEg95 to obtain the recombinant secreted plasmid pSMEg95. The recombinant plasmids were then transformed to BCG and M. smEgmatis through electroporation respectively. It was demonstrated through double restriction endonuclease digstion, PCR amplification and gene sequencing that the Eg95- coding gene sequence and the BCG- Ag85B signal sequence were successfully inserted to plasmid pMV261, indicating that the recombinant secreted and non-secreted BCG-Eg95 and M. smegamatis-Eg95 vaccine for E. granolosus have been successfully constructed.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2008年第6期526-530,共5页
Chinese Journal of Zoonoses
基金
甘肃省自然科学基金(No.3ZS051-A25-080)
国家科技部863项目(No.2006AA02Z420)部分资助
