小鼠BPI_(36-259)抗菌蛋白在原核细胞中的表达及其抗血清的制备  

Expression of muBPI_(36-259) Recombinant Protein Prokaryotic Expression Vector and Preparation of a Polyclonal Antiserum Against muBPI_(36-259) Product

作　　者：冯颖[1] 李丽[1] 龙军[1] 范宜强[1] 刘振龙[1] 孔庆利[1] 吕喆[1] 王炜[1] 安云庆[1] 

机构地区：[1]首都医科大学基础医学院免疫学系

出　　处：《首都医科大学学报》2008年第2期184-189,共6页Journal of Capital Medical University

摘　　要：目的采用原核表达系统获得小鼠BPI36-259目的抗菌蛋白,免疫家兔制备目的蛋白特异性抗血清。方法利用生物信息学方法预测小鼠BPI功能片段,构建pUC57-muBPI1-281质粒;PCR扩增获得mu BPI36-259基因片段,构建pET28a-mu BPI36-259重组质粒;用IPTG诱导重组质粒转化的E.coli BL21(DE3)感受态细胞,获得以包涵体形式表达为主的mu BPI36-259目的蛋白;用变性目的蛋白免疫家兔获得兔抗鼠BPI36-259蛋白特异性抗血清;用间接ELISA法检测血清抗体特异性及其效价。结果成功构建了pET28a-mu BPI36-259重组质粒,转化E.coli BL21(DE3)获得以包涵体表达形式为主的目的蛋白;用变性目的蛋白免疫家兔获得小鼠BPI36-259蛋白特异性抗血清(效价1∶10000)。结论在原核细胞中成功获得了以包涵体表达形式为主的抗菌蛋白,免疫家兔获得的特异性抗血清可用于小鼠BPI36-259目的抗菌蛋白的检测。Objective To make murine muBPI36-259 protein with prokaryotic expressing system and prepare rabbit polyclonal antiserum with the recombinant protein. Methods Functional domain of murine BPI was predicted by bioinformatics methods, cDNA of muBPI1-281 was synthesized and cloned to pUC57 cloning vector to generate pUC57-muBPI1-281 construct. Then muBPI36-259 functional fragment was amplified on the pUC57-muBPI1-281 plasmid by PCR and cloned into prokaryotic expression vector, pET28a, to produce the expression vector pET28a-muBPI36-259. After IPTG inducing, recombinant protein was expressed in bacteria E. coli BL21 （DE3） in the main form of inclusion bodies. This recombinant protein was used to immunize rabbit to generate polyclone anti - serum, the titer and the specificity of antiserum was detected by indirect ELISA method. Results Prokaryotic expression construct at murine BPI functional domain（pET2ga-muBPI36-259） has been successfully generated, which express this recombinant protein in the main form of inclusion bodies. Using this recombinant protein to immunize rabbit, we got specific anti-mumuBPI36-259 antiserum with the titer of 1：10 000. Conclusion We have obtained the antibiosis protein （ muBPI36-259 ） in main form of inclusion bodies from prokaryotic expression system and used it to immunize rabbit, the specific antiserum can be used to detect the target antibiosis protein of muBPI36-259.

关 键 词：小鼠BPI36-259目的蛋白 E.COLI BL21(DE3) 多克隆抗血清 

分 类 号：R587.1[医药卫生—内分泌]