摘要
用大肠杆菌-枯草芽孢杆菌穿梭载体pNW33N和去除了信号肽编码序列的成熟mpd基因构建了穿梭启动子探针pNW33N-mpd。用该探针从质粒pMPDP3和pMPDP29上克隆来自于枯草芽孢杆菌ytkA和ywoF基因上游的启动子功能片段,构建了穿梭表达载体pNYTM和pNYWM。将表达载体pNYTM和pNYWM转入枯草芽孢杆菌1A751获得表达菌株1A751(pNYTM)和1A751(pNYTM),mpd基因在ytkA和ywoF基因的启动子和信号肽的带动下实现了分泌表达且具有天然活性,结果表明ytkA基因的启动子强度强于ywoF基因的启动子。利用ytkA基因的强启动子和nprB基因的分泌型信号肽编码序列构建了新的穿梭分泌表达载体pYNMK,并使mpd基因在枯草芽孢杆菌WB800中得到了更高水平的分泌表达,表达菌株WB800(pYNMK)在培养到第84h时甲基对硫磷水解酶酶活达到最高值为10.40u/mL,是出发菌株邻单胞菌M6表达量的10.8倍,重组表达产物有91.4%分泌在培养基中。
A shuttle promoter-probe vector pNW33N-mpd was constructed with the E. coli-B, subtilis shuttle vector pNW33N and the mature mpd gene without it＇ s signal peptide-encoding sequence. The promoter fragments of B. subtilis ytkA and ywoF gene were cloned from plasmid pMPDP3 and pMPDP29 then generated the shuttle expression vector pNYTM and pNYWM. Expression vectors pNYTM and pNYWM were transformed into B. subtilis 1A751 to construct the expression strain 1A751 （pNYTM） and 1A751 （pNYTM）, in these strains, under the control of the promoters and signal peptides of ytkA and ywoF gene, mpd gene was expressed and secreted with it＇ s biological activity ; the result showed that the promoter of ytkA gene is much stronger than that of ywoF gene. Then a new shuttle expression-secretion vector pYNMK was constructed using the ytkA gene promoter and the signal peptide-encoding sequence of B. subtilis nprB gene, the expression of mpd gene achieved a higher level using the B. subtilis WB800 as the host, the methyl parathion hydrolase activity accumulated to a maximum level of 10.40 u/mL after 84 h of cultivation at the late stationary phase, which was 10.8-fold higher than the expression level of the original Plesiomonas strain M6, about 91.4% of the recombinant expression production was secreted into the culture medium.
出处
《生物工程学报》
CAS
CSCD
北大核心
2006年第2期249-256,共8页
Chinese Journal of Biotechnology
基金
国家自然科学基金(No.30300005)
国家"863计划"(No.2004246070
2004214102)
国家科技攻关项目(No.2005BA516A02-01
2005BA516A02-02)资助。~~
