摘要
目的 :采用巴斯德毕赤酵母表达系统表达分泌型功能性rBPIm2 3抗菌蛋白。方法 :利用构建的pUC1 8 synBPI4 1 4质粒和PBV2 2 0 BPIm4 2 0 质粒 ,按分子克隆方法构建pPICZα synBPIm6 0 0 重组表达载体 ;用线性化pPICZα synBPIm6 0 0 质粒电转化PichiapastorisGS1 1 5 ,筛选抗性转化子 ,进行PCR和Mut表型鉴定 ;用甲醇诱导目的蛋白rBPIm2 3的表达 ;用离子交换层析纯化rBPIm2 3,并进行SDS PAGE分析、Westernblot鉴定和用BCA法定量。结果 :①成功构建了pPICZα synBPIm6 0 0 重组表达载体 ;②获得稳定整合目的基因的GS1 1 5菌株 ;③表达产物相对分子量约为 2 3kD ,可与抗BPI抗体特异性结合 ;④培养上清液中目的蛋白表达量约为 2 9mg L。结论 :BPIm2 3重组抗菌蛋白在毕赤酵母GS1 1 5中得到分泌表达。
Objective:To express and secrete rBPI m23 bactericidal protein in Pichia pastoris expression system. Methods:pPICZα synBPI m600 recombinant expression vector was constructed with prepared vectors (pUC18 synBPI 414 and PBV220 BPI m420 ) according to Molecular Cloning Laboratory Manual. The Pichia pastoris GS115 were electroporated with linearized pPICZα synBPI m600 vectors , positive clones were screened on low salt LB medium with Zeocin , then the positive clones were identified by PCR and Mut phenotype analysis. rBPI m23 was induced to express in BMMY ,purified by SP Sepharose beads , identified by SDS PAGE and Western blot and measured with BCA method. Results:①pPICZα synBPI m600 expression vector was successfully constructed.②Pichia pastoris GS115 strains integrated with synBPI m600 stably were obtained.③SDS PAGE showed the molecular weight of the recombinant protein was approximate 23 kD,and Western blot confirmed that the protein could bind to the anti BPI antibody specially.④The supernate protein concentration was about 2 9 mg/L.Conclusion:rBPI m23 was expressed and secreted effectively in Pichia pastoris GS115.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2004年第6期374-378,共5页
Chinese Journal of Immunology
基金
国家自然科学基金项目资助 (编号 3 95 70 666)
