利用短短小芽孢杆菌启动子和信号肽编码序列构建穿梭分泌表达载体

以短短小芽孢杆菌B15的总DNA为模板,利用PCR技术克隆到其细胞壁蛋白基因串联启动子和信号肽编码序列,测序分析后提交GenBank,登录号为AY956423。重新设计引物扩增该片段并在PCR产物两侧引入BamHⅠ和PstⅠ酶切位点,将PCR产物双酶切后克隆至穿梭载体pP43NMK的相应位点构建分泌表达载体pP15MK,插入片段置于该载体中mpd基因的上游,并使信号肽编码序列与去除了自身信号肽编码序列的mpd基因阅读框恰好融合。将pP15MK导入枯草杆菌构建表达菌株1A751(pP15MK),在短短小芽孢杆菌启动子和信号肽元件的带动下,mpd基因能够在表达菌株的对数生长期和稳定期持续性高效分泌表达,表达产物结合在细胞膜上;发酵液在48h酶活达到最高值7.79U/mL,是出发菌株邻单胞菌M6表达量的8.1倍。

Construction of the shuttle expression-secretion vector with the promoters and signal peptide-encoding sequence from Brevibacillus brevis

The multiple and tandem promoters and signal peptide-encoding sequence of cell wall protein encoding gene was amplified from Brevibacillus brevis B15 total DNA,the PCR fragment was cloned,sequenced and analyzed,then was submitted to GenBank with a Accession No.AY956423.Another pair of primers were designed to amplify the fragment again,BamHI and PstI sites was introduced flanking the PCR production.BamHI/PstI digested fragment was cloned into the corresponding site of shuttle vector pP43NMK to generate the expression-secretion vector pP15MK.The inserted fragment was upstream of mpd gene and the signal peptide-encoding sequence was fused in frame with the mpd gene,which its own signal peptide-encoding sequence was deleted.The recombinant vector was transformed into Bacillus subtilis 1A751,under the control of the promoters and signal peptide from Brevibacillus brevis B15,mpd gene was continuously expressed and secreted at a high efficiency throughout the exponential growth phase and into the late stationary phase,the expression production methyl parathion hydrolase(MPH) was attached on the outside of the cell membrane.MPH activity accumulated to a maximum level of 7.79 U/mL after 48 h of cultivation at the late stationary phase,which was 8.1-fold higher than the expression level of the original Plesiomonas strain M6.