基于冰核蛋白的乳酸菌表面展示系统的构建

Construction of cell surface display system in lactic acid bacteria by using ice nucleation protein

【目的】验证来源于丁香假单胞菌的冰核蛋白在乳酸乳球菌表面展示外源蛋白的可能性。【方法】以绿色荧光蛋白(Green Fluorescence Protein,GFP)基因gfp为报告基因,以冰核蛋白基因的N末端和NC端作为展示单元,构建乳酸菌表面展示载体pHZ101和pHZ102,并转化大肠杆菌(Escherichia coli)JM109和乳酸乳球菌(Lactococcus lactis)MG1363。【结果】荧光显微镜观察显示重组大肠杆菌和乳酸乳球菌均能检测到绿色荧光。Western blot结果表明GFP蛋白在重组大肠杆菌和乳酸乳球菌中均得到表达,并且INPN-GFP蛋白多数滞留于乳酸乳球菌细胞质内,而INPNC-GFP蛋白则大部分定位于乳酸乳球菌的细胞膜上。【结论】以上结果表明丁香假单胞菌的冰核蛋白能引导外源蛋白定位于乳酸乳球菌的细胞膜上,为乳酸菌表面展示系统的构建提供了新的方向。

[Objective]The gene of ice nucleation protein （INP）,an outer membrane of Pseudomonas syringae was tested to display foreign proteins on the surface of Lactococcus lactis.[Methods] Plasmids pHZ101 and pHZ102 were constructed using gfp （Green Fluorescence Protein） as the reporter gene and N-terminal and NC-terminal of inp as the anchoring units. Plasmids pHZ101 and pHZ102 were subsequently transformed into Escherichia coli JM109 and Lactococcus lactis MG1363.[Results] Fluorescence microscope shows that green flrorescence was observed in both recombinant E.coli and L. lactis strains. Western blot indicated that GFP was expressed in both recombinant E.coli and L.lactis strains. INPN-GFP was mostly trapped in cytoplastic fraction while INPNC-GFP was mainly targeted on the cell membrane of L.lactis.[Conclusion]The results suggest a new way to construct cell surface display system of lactic acid bacteria by using ice nucleation protein.