摘要
选取金黄色葡萄球菌（Staphylococcus aureus）的Ebps和ClfA、大肠杆菌（Escherichia coli）的OmpA和OmpC、无乳链球菌（Streptococcus agalactiae）的SIP和PGK 3种常见致病菌的特异性蛋白。通过分析3种致病菌的B/T细胞优势蛋白表位并预测和评价其抗原性,设计合成三联重组表位序列（TRE）。应用腺病毒Ad-Max系统将合成后的重组表位序列连接到穿梭载体pDC315-MCS-EGFP上,与腺病毒大骨架pBHGloxE1、3Cre共转染HEK293细胞,1周后观察到转染细胞出现绿色荧光且细胞出现典型的CPE,证明穿梭质粒载体pDC315-MCS-EGFP和腺病毒大骨架pBHGloxE1、3Cre转染成功。RT-PCR、Western blot验证所获得的重组腺病毒Ad-TRE-EGFP,PCR鉴定P4代重组腺病毒,均得到目的条带。说明目的片段在HEK293细胞中稳定有效表达。为下一步3种常见致病菌重组表位疫苗的体内外试验奠定了工作基础。
The specific proteins of three common pathogens were selected, Ebps and ClfA of Staph- ylococcus aureus, OmpA and OmpC of Escherichia coli, SIP and PGK of Streptococcus agalactiae. By analysising of three common pathogens in B/T cell dominant epitopes,predicting and evalua- ting its antigenicity, the triple recombinant epitopes sequence（TRE）were designed and synthe- sised. Application of adenovirus Ad Max system,connecting the recombinant epitopes sequence to pDC315-MCS-EGFP,and HEK293 cells were co transfected with pBHGloxE1,3Cre. After one week, the transfected cells displayed green fluorescence and typical CPE, which showed that pDC315-MCS-EGFP and pBHGloxE1,3Cre transfection were successfully constructed. RT-PCR and Western blot were used to identify the Ad TRE-EGFP, the generation of P4 Ad-TRE-EGFP was identified by PCR. All obtained the target strip. The result indicated that the target fragment was stable and effective in HEK293 cells,which laid the foundation for the further study of the re- combinant epitope vaccine of three common pathogenic bacteria in vitro and in vivo.
作者
林静
尚翠玲
李小慧
高珂珂
孟佳丽
李昕
金天明
LIN Jing,SHANG Cui-ling,LI Xiao-hui,GAO Ke-ke,MENG Jia li,Li Xin,JIN Tian-ming(College of Animal Science and Animal Medicine, Tianjin Agriculture University, Tianjin 300384 ,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2018年第11期2094-2099,共6页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(31572492)
天津市优秀科技特派员资助项目(16JCTPJC45000)
天津市自然科学基金重点资助项目(12JCZDJC22100)
天津市农委农业科技示范推广资助项目(201202110)
天津市兽医生物技术优秀科研创新团队资助项目(TD12-5019)
天津市兽医学重点学科“领军人才培育计划”人选资助项目
关键词
金黄色葡萄球菌
大肠杆菌
无乳链球菌
重组表位
重组腺病毒
Staphylococcus aureu
Escherichia coli
Streptococcus agalactiae
recombinant epitopes
recombinant adenovirus
