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目的构建可以在双歧杆菌表达外源性基因的系统。方法 PCR扩增双歧杆菌质粒聚合酶基因(BPP)并连接至质粒pBS-T以形成重组质粒pBS-BPP。PCR扩增双歧杆菌内源性阿拉伯糖苷酶的启动子及分泌性信号肽DNA序列(ara)并连接至质粒pBAD-A以形成重组质粒pBAD-ara,然后将增强绿色荧光蛋白(eGFP)基因连接至质粒pBAD-ara以形成重组质粒pBAD-ara-GFP。采用基因重组技术重组含有ara、BPP、eGFP基因序列并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pBS-BPP-ara-GFP,激光共聚焦显微镜下观察含有pBS-BPP-ara-GFP质粒及对照质粒的E.coli,验证eGFP定位表达情况。结果所构建的表达系统可以在E.coli中表达eGFP基因。结论通过基因重组方法成功构建了双歧杆菌表达系统,其可将外源基因分泌表达于菌体外。
Objective To construct a system that can express foreign genes in Bifidobacterium. Methods Bifidobacterium plasmid polymerase gene (BPP) was amplified by PCR and ligated into plasmid pBS-T to form recombinant plasmid pBS-BPP. The promoter of Bifidobacterium endogenous arabinosidase and the secretory signal peptide DNA sequence (ara) were PCR-amplified and ligated into the plasmid pBAD-A to form the recombinant plasmid pBAD-ara, and then the green fluorescent protein (eGFP) gene Ligated to plasmid pBAD-ara to form recombinant plasmid pBAD-ara-GFP. The recombinant plasmid pBS-BPP-ara-GFP containing ara, BPP and eGFP gene was cloned by gene recombination technique. The plasmid pBS-BPP-ara-GFP secreting exogenous gene was expressed in vitro and anchored to the cell wall. -BPP-ara-GFP plasmid and control plasmid E.coli verify eGFP localization expression. Results The constructed expression system can express eGFP gene in E. coli. Conclusion The Bifidobacterium expression system was successfully constructed by gene recombination, which secreted exogenous genes into the outside of mycelium.