利用细胞壁锚定蛋白MbA构建新型苏云金芽胞杆菌表面展示系统  

Development of a novel cell-surface display system in Bacillus thuringiensis using cell wall-binding protein MbA as the anchoring motif

作　　者：吕婷[1] 沈霏[1] 邵小虎[1] 李华[1] 江梦天[1] 李林[1] 

机构地区：[1]华中农业大学生命科学技术学院/农业微生物学国家重点实验室,武汉430070

出　　处：《华中农业大学学报》2014年第1期7-11,共5页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(30670054;31070111)

摘　　要：从苏云金芽胞杆菌(Bacillus thuringiensis)野生型菌株YBT-9602基因组中通过PCR扩增到编码一种具有细胞壁锚定活性的芽胞皮层水解酶的编码基因mbA。序列测定和编码产物结构域预测分析显示,mbA编码产物在结构上由1个具有肽聚糖结合性能的N-末端结构域和1个具细胞壁水解酶活性的C-末端结构域组成,具有作为细胞表面展示系统的运载蛋白的潜力。通过体外构建mbA与绿色荧光蛋白基因gfp的融合基因(mbA-gfp)并导入苏云金芽胞杆菌受体菌中进行表达,经对重组菌全细胞免疫荧光显微镜观测、蛋白酶pronase消化试验和SDS处理,证实GFP被成功展示于受体菌细胞表面;经流式细胞仪测定,以细菌β-1,3-1,4-葡聚糖酶为靶蛋白的表面展示系统有42.97%的重组菌细胞可展示融合酶,重组菌具有13.5U/mL的全细胞酶活性。结果表明,MbA作为一种细胞壁锚定蛋白可用于构建新的苏云金芽胞杆菌细胞表面展示系统。A Bacillus spore cortex-lytic enzyme with the cell wall-binding activity, MbA,was used to develop a novel cell-surface display system of Bacillus thuringiensis. The full-length of tuba gene from B. thuringiensis wild type strain YBT-9602 genome was PCR-amplified, sequenced and the struc- tural features of its encoding domains were characterized. It showed that the predicted protein MbA was structurally distinguished by an N terminal domain with peptidoglycan binding activity and a C-terminal domain with cell wall hydrolysis activity. By constructing and expressing the fused mbA-gfp, it showed that the heterologous GFP was successfully immobilized onto the surface of B. thuringiensis cells through the immunofluorescence microscopic observation and the assays of pronase accessibility and SDS sensitivity. Furthermore, the recombinant B. thuringiensis cells expressing the MbA fused chimeric pro- tein with a bacterial β 1,3-1,4-glucanase, were determined the surface-display efficiency using flow cy tometry analysis,which showed that a 42.97% recombinant cells exhibited surface displaying activity. The whole-cell enzymatic activity of the recombinant B. thuringiensis MB220 expressing the rnbA-glS fusion gene was detected to be 13.5 U/mL. These results indicated that the MbA protein can be served as a functional carrier protein for developing a novel B. thuringiensis cell surface display system.

关 键 词：苏云金芽胞杆菌 表面展示系统 细胞壁锚定蛋白 芽胞皮层溶解酶 绿色荧光蛋白基因 

分 类 号：Q939.9[生物学—微生物学]