Construction of a Shuttle Vector for Protein Secretory Expression in Bacillus subtilis and the Application of the Mannanase Functional Heterologous Expression
Construction of a Shuttle Vector for Protein Secretory Expression in Bacillus subtilis and the Application of the Mannanase Functional Heterologous Expression
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枯草芽孢杆菌蛋白分泌表达穿梭载体的构建及甘露聚糖酶功能异源表达的应用
DOI：
10.4014/jmb.1311.11009
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发表时间：
2014-04-01
影响因子：
2.8
通讯作者：
Wei, Wei
中科院分区：
文献类型：
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作者：
Guo, Su;Tang, Jia-jie;Wei, Wei
我们报道了两个枯草芽孢杆菌表达载体pBNS 1/pBNS 2的构建。两种载体均基于强启动子P43和氨苄青霉素抗性基因表达盒。此外，插入具有Shine-Dalgarno序列和多克隆位点（BamHI、SalI、SacI、XhoI、Pst I、SphI）的片段。将amyQ（编码淀粉酶）信号肽的编码区与pBNS 1的启动子P43融合，构建分泌型表达载体pBNS 2。通过首先产生表达载体pBNS 1-GFP/pBNS 2-GFP，然后检测绿色荧光蛋白基因表达来测试载体的适用性。接下来，来自B的甘露聚糖酶基因。pumilus Nsic-2与载体pBNS 2融合，并测定了上清液中甘露聚糖酶的活性。甘露聚糖酶总酶活为8.65 U/ml，比出发菌株提高了6倍。我们的工作为在B中建立有效的基因表达转化体系提供了一条可行的途径。枯草芽孢杆菌（B. subtilis），并首次报道获得B.短小甘露聚糖酶在B中的分泌表达。枯草杆菌。
We report the construction of two Bacillus subtilis expression vectors, pBNS1/pBNS2. Both vectors are based on the strong promoter P43 and the ampicillin resistance gene expression cassette. Additionally, a fragment with the Shine-Dalgarno sequence and a multiple cloning site (BamHI, SalI, SacI, XhoI, PstI, SphI) were inserted. The coding region for the amyQ (encoding an amylase) signal peptide was fused to the promoter P43 of pBNS1 to construct the secreted expression vector pBNS2. The applicability of vectors was tested by first generating the expression vectors pBNS1-GFP/pBNS2-GFP and then detecting for green fluorescent protein gene expression. Next, the mannanase gene from B. pumilus Nsic-2 was fused to vector pBNS2 and we measured the mannanase activity in the supernatant. The mannanase total enzyme activity was 8.65 U/ml, which was 6 times higher than that of the parent strain. Our work provides a feasible way to achieve an effective transformation system for gene expression in B. subtilis and is the first report to achieve B. pumilus mannanase secretory expression in B. subtilis.