摘要
目的构建可以在乳链菌表达外源性基因的系统。方法采用基因拼接方法拼装含有P59启动子、USP45蛋白信号肽及多克隆位点的基因序列,并将其连入质粒pBluescriptIISK(+)以形成重组质粒pBS-PU,PCR扩增USP45终止子并连接至质粒pBS-PU构建可将外源性基因分泌表达于菌体外的质粒pNBC1000,PCR扩增M6基因随后连接至pNBC1000构建可将外源性基因分泌表达于菌体外及锚定表达于细胞壁的质粒pNBC2000。通过将增强绿色荧光蛋白(EGFP)基因连接至质粒pNBC2000,激光共聚焦显微镜下观察含有pNBC2000-EGFP质粒及对照质粒的细菌,验证EGFP定位表达情况。结果所构建的表达系统可以表达EGFP基因。结论通过基因拼接方法成功构建了乳链菌表达系统,其可将外源性基因分泌表达于菌体外。
Objective To construct and characterize an exogenous gene expression system in Lactococcus lactis. Methods PCR-based gene assembly was used to synthesize the gene sequence containing P59 promoter, USP45 signal peptide, ribosome binding site and multiple cloning site. Plasmid pBS-pu was obtained after ligation of the assembled sequence with pBluescript Ⅱ SK （＋）, and the terminator of USP45 protein was added to construct the recombinant plasmid pNBC1000. The gene coding for Streptococcus pyogenes M6 protein was amplified and cloned into pNBC1000 to obtain the plasmid pNBC2000. To characterize the expression system, enhanced green fluorescent protein （EGFP） gene was amplified fi＇om the plasmid pEGFP-Nl and cloned into pNBC2000. Laser scanning confocal microscope was used to observe the bacteria containing pNBC2000, pNBC2000-EGFP or pBS-EGFP. Results Green fluorescence wad wisualized in the bacteria containing pNBC2000-EGFP, but not in the bacteria containing pNBC2000 or pBS-EGFP. Conclusion The plasmids pNBC1000 and pNBC2000 containing P59 promoter, USP45 signal peptide, ribosome binding site and multiple cloning sites are successfully constructed, which are capable of expressing exogenous gene in Lactococcus lactis.
出处
《第一军医大学学报》
CSCD
北大核心
2005年第10期1232-1235,1239,共5页
Journal of First Military Medical University
基金
国家自然科学基金(30570839)
广东省自然科学基金(05004735)
广州市科委基金(2002Z3-E4101)~~
关键词
基因拼接
乳链菌
外源性基因
表达系统
gene assembly
Lactococcus lactis
exogenous gene
expression system
