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《Chinese Journal of Microecology》 2015-01
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Construction and identification of constitutive surface display vector on Lactococcus lactis

FENG Meng-die;MAO Pu-jia;HONG Yu;MAO Xiao-ping;XU Ze-yang;SONG Wu-zhan;YANG Hong-wen;ZHAO Ji-hua;HUANG Fen;JING Shen-rong;ZENG Wei-kun;Medical Faculty,Kunming University of Science and Technology;Department of Nuclear Medicine,Kunming General Hospital of Chengdu Military Command;Department of Urology,Central Hospital of Xiaogan;Medical Faculty,Kunming University;  
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.
【Fund】: 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)
【CateGory Index】: Q78
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