乳酸乳球菌组成型表面展示载体的构建及鉴定  

Construction and identification of constitutive surface display vector on Lactococcus lactis

作　　者：冯梦蝶[1] 毛普加 洪愉[2] 毛小萍[3] 许泽仰 宋武战[2] 杨洪文[2] 赵继华[2] 黄芬[1] 井申荣[1] 曾韦锟[1,4] 

机构地区：[1]昆明理工大学医学院,云南昆明650500 [2]成都军区昆明总医院核医学科,云南昆明650032 [3]孝感市中心医院泌尿外科,湖北孝感432000 [4]昆明学院医学院,云南昆明650214

出　　处：《中国微生态学杂志》2015年第1期1-5,共5页Chinese Journal of Microecology

基　　金：国家自然科学基金(31160193);云南省教育厅科学研究基金(2010Y398);云南省应用基础研究面上项目(2010ZC055;2012FB135)

摘　　要：目的探索融合有Usp45信号肽和3Lys M锚定序列的EGFP蛋白能否通过p32启动子组成型展示于乳酸乳球菌MG1363表面。方法融合PCR法分别将p32启动子与Usp45信号肽、3Lys M与egfp基因融合亚克隆至p MD-18T载体,鉴定正确后命名为18T-p Usp45、18T-3Lys M-egfp,分别将上述片段切下依次插入p MG36e构建p MG36e-p Usp45-3Lys M-egfp。将其转化入乳酸乳球菌MG1363,荧光显微镜观察及SDSPAGE、Western-blot检测。结果成功构建重组菌p MG36e-p Usp45-3Lys M-egfp/MG1363,荧光显微镜、SDSPAGE和Western-blot检测表明重组蛋白能在MG1363中组成型表达,且分泌至胞外并锚定在细胞壁上。结论成功地构建了组成型表面展示载体p MG36e-p Usp45-3Lys M-egfp,重组蛋白能分泌至胞外且展示在MG1363细胞壁上。Objective To explore whether EGFP protein fused with Usp45 signal peptide and 3Lys M anchor sequencer could be constitutively displayed on the surface of Lactococcus lactis MG1363 by p32 promoter. Methods The exogenous gene with anchor sequence（ 3Lys M-egfp） and Usp45 signal peptide with p32 promoter were obtained by fusion PCR,respectively. They were subcloned into p MD18-T vector for sequencing,which was named18T-3Lys M-egfp and 18T-p Usp45,respectively. The above fragments were inserted into p MG36 e for construction of the constitutive expression vector p MG36e-p Usp45-3Lys M-egfp. Then the vector was transformed into Lactococcus lactis MG1363. Fluorescence microscopy,SDS-PAGE and Western-blot were used to analyze the expression of the fusion protein in MG1363. Results Recombinant constitutive expression strain p MG36e-p Usp45-3Lys M-egfp /MG1363 was successfully constructed. Fluorescence microscopy,SDS-PAGE and Western-blot analysis showed that the recombinant protein could be constitutively secreted and anchored on MG136 cell walls. Conclusion The constitutive surface display vector p MG36e-p Usp45-3Lys M-egfp was constructed successfully,and the recombinant protein could be secreted and anchored on MG1363 cell walls.

关 键 词：乳酸乳球菌 表面展示 锚定 组成型表达 

分 类 号：R378.992[医药卫生—病原生物学]