产单核细胞李斯特菌p60蛋白N端肽聚糖结合基序在枯草芽孢杆菌中的表达  

Expression of N-Terminal Peptidoglycan Binding Motif of Extracellular Protein p60 from Listeria monocytogenes in Bacillus subtilis

作　　者：江巍 袁利思 陶好霞[2] 关清 刘纯杰[2] 赵宝华[1] 王艳春[2] JIANG Wei;YUAN Li-Si;TAO Hao-Xia;GUAN Qing;LIU Chun-Jie;ZHAO Bao-Hua;WANG Yan-Chun(School of Life Science, Hebei Normal University, Shijiazhuang 050024,China;Beijing Institute of Biotechnology, Beijing 100071, China)

机构地区：[1]河北师范大学生命科学学院,河北石家庄050024 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2017年第6期731-736,共6页Letters in Biotechnology

摘　　要：目的:构建产单核细胞李斯特菌胞外蛋白p60的N端肽聚糖结合基序(p60-N)表达载体,实现其在枯草芽孢杆菌中的分泌表达。方法:将目标基因克隆到表达载体pHT43中获得重组载体pHT43-p60-N,转化枯草芽孢杆菌WB800N获得重组工程菌株WB800N/pHT43-p60-N,在此基础上考察IPTG浓度、培养基、表达时间和温度等条件对目标蛋白表达的影响。结果:p60-N蛋白可在枯草芽孢杆菌中分泌表达,与鼠李糖乳杆菌形成的革兰阳性增强基(GEM)颗粒特异性结合,具有与天然蛋白类似的生物学活性。用GB培养基,在37℃下,加入终浓度为0.2 mmol/L的IPTG诱导12~20 h,表达量提高到28 mg/L。结论:实现了p60蛋白N端基序在枯草芽孢杆菌中的分泌表达,为进一步研究以该蛋白为基础的细菌样颗粒疫苗奠定了基础。To epxress the N-terminal peptidoglycan binding motif of extracellular protein p60(p60-N)produced by Listeria monocytogenes in Bacillus subtilis WB800N.Methods:The target gene was inserted into vector pHT43to construct a recombination plasmid pHT43-p60-N,which was subsequently transformed into WB800N to produce engineering strain WB800N/pHT43-p60-N.Then the expression conditions were investigated,including IPTG concentration,culture medium,expression time and temperature.Results:The protein of p60-N was expressed in B.subtilis,and it specifically bound to Gram-positive enhancer matrix(GEM)particles formed by Lactobacillus rhamnosus,showing similar biological activity with its natural counterparts.In GB medium,IPTG was added to a final concentration of0.2mmol/L for induction of the target protein within12~20h at37℃,as a result,the expression quantity of target protein was increased to28mg/L.Conclusion:The soluble and active p60-N protein was obtained using B.subtilis secretion expression system,which will be used for development of the bacteria-like particle vaccines.

关 键 词：p60蛋白N端肽聚糖结合基序 枯草芽孢杆菌 革兰阳性增强基-蛋白锚钩(GEM-PA)表面展示系统 

分 类 号：Q78[生物学—分子生物学]