小鼠BPI N端功能基因片段的克隆及其表达产物的胞内杀菌作用  

Cloning of N-terminal functional gene fragment of murine BPI gene and studies on its intracellular bacteriocide function

作　　者：李丽[1] 冯颖[1] 吕喆[1] 庆利[1] 刘振龙[1] 赵冬[1] 高燕[1] 王炜[1] 安云庆[1] 

机构地区：[1]首都医科大学免疫学系,北京100069

出　　处：《中国免疫学杂志》2008年第6期500-503,508,共5页Chinese Journal of Immunology

基　　金：北京市自然科学基金项目(7082016)资助

摘　　要：目的:克隆小鼠BPI36-259基因片段,转染细胞获得具有杀菌作用的目的抗菌蛋白。方法:采用生物信息学方法,构建PUC57-muBPI1-281质粒;PCR扩增获得muBPI36-259基因片段,制备pcDNA3.1(+)-muBPI36-259重组质粒;采用电穿孔法将上述重组质粒转染RAW264.7细胞,Western blot法检测目的蛋白在胞内的表达;建立胞内杀菌实验模型,检测muBPI36-259目的蛋白对胞内寄生菌(伤寒杆菌)的杀伤作用。结果:muBPI36-259目的蛋白在RAW264.7细胞内成功表达,对胞内寄生菌-G-伤寒杆菌具有杀伤作用。结论:成功克隆了小鼠BPI36-259基因片段,转染RAW264.7细胞获得具有生物学活性目的抗菌蛋白。Objective： To clone the N-terminal fragment of mouse BPI from 36 to 259 amino acid and to identify the target protein for bacteriocide function by transfected into RAW264.7 cells. Methods： PUC57-muBPI1-281 plasmid was synthesized by comparision to human BPI N-terminal functional fragment. Then an encoding fragment for functional mouse BPI36-259 was obtained by PCR The fragment was cloned in expressing vector pcDNA3.1 （ ＋ ） to construct pcDNA3.1 （ ＋ ） - muBPI36-259 recombinant plasmid. RAW264.7 cells were transfected using electroporation method and the expression of target protein was detected by Western blot. Furthermore, bacteriocide function of the recombinant protein to intracellular Salmonella typhi was identified. Results： The target protein of muBPI36-259 was successftdly expressed in RAW264.7 cells, and it could kill the intracellular G^- Salmonella typhi. Conclusion： The gene fragment coding for mouse BPI36-259 has successfully been cloned and transfeeted into RAW264.7cells. The target antibacterial protein exhibit bacteriocide function against intracellular Salmonella typhi.

关 键 词：杀菌/渗透性增强蛋白 RAW264.7细胞 伤寒杆菌 

分 类 号：R392[医药卫生—免疫学]