摘要
【目的】本试验旨在筛选引导表达外源木聚糖酶基因高效分泌的信号肽,为枯草芽胞杆菌木聚糖酶高效分泌表达系统提供元件。【方法】构建信号肽筛选载体,载体是以含壮观霉素抗性基因的大肠-枯草穿梭载体为基本骨架,目标蛋白为耐碱性木聚糖酶,可在麦芽糖启动子Pglv诱导下表达。从枯草芽胞杆菌A1747基因组中扩增获得24个Sec途径信号肽,并将其全部链接到至筛选载体上,并在枯草芽胞杆菌WB700中实现表达分泌。重组菌在3%麦芽糖诱导下培养24h后用DNS法测定上清酶活。【结果】成功构建信号肽筛选载体pGPSX及24个表达载体,实现木聚糖酶表达分泌。且不同信号肽对于引导外源木聚糖酶分泌能力不同,其中YnfF信号肽引导分泌目标蛋白效率最高,上清酶活为37.2IU/mL。【结论】试验证明在枯草杆菌中对外源蛋白进行信号肽筛选是提高其分泌的有效途径,并获得了针对木聚糖酶高效分泌信号肽YnfF。
[Objective] We searched optimal signal peptide for heterologous and exogenous secretion of xylanase in Bacillus subtilis.[Methods] We constructed a screening vector for signal peptides from B.subtilis.The Alkali resistance xylanase gene（xynA） from Bacillus pumilus was chosen as reporter gene and cloned into E.coli and B.subtilis shuttle vector pGJ148 which has maltose-inducible promoter Pglv and spectinomycin resistant gene.24 Sec-type signal peptides（SPs） was amplified from B.Subtilis 1A747 and cloned into the screening vector for the expression of xynA in B.Subtilis WB700.The xylanase activity of the culture supernatant were detected after 24h incubation.[Results] The screening of these signal peptides revealed differences in xylanase activity of the culture supernatants,The recombinant strain containing YnfF signal peptide showed the highest xylanase acitivity（37.2IU/mL）.[Conclusion] Experiment proved screening of signal peptides is effective way for optimization of the export of heterologous protein in B.subtilis.
出处
《微生物学报》
CAS
CSCD
北大核心
2011年第7期979-983,共5页
Acta Microbiologica Sinica
基金
国家自然科学基金(30871813)~~
