双功能枯草杆菌诱导型高效表达分泌载体的构建与鉴定

利用大肠杆菌质粒pSP72和枯草杆菌质粒pUB18共整合得到双功能克隆载体pSB。在pSB多克隆位点依次引入枯草杆菌果聚糖蔗糖酶基因启动子-信号肽序列sacBp.s.、地衣芽孢杆菌淀粉酶基因终止子序列α-amyT和短小芽孢杆菌增强子基因degQ,最终构建了双功能枯草杆菌诱导型高效表达分泌载体pSBPTQ。将VasostatinⅠ基因作为靶基因检测sacBp.s.、α-amyT和degQ在pSBPTQ进行外源基因表达时的功能,结果表明,在蔗糖诱导下,sacB启动子有效启动了Vasostatin I基因的表达和分泌,α-amy T提高了VasostatinⅠ基因的转录效率,而degQ明显增强了VasostatinⅠ基因的表达水平。VasostatinⅠ基因在蔗糖诱导下成功表达并分泌到枯草杆菌细胞外,蛋白质分泌效率达到90%左右。质粒稳定性试验结果表明,经过40个世代之后,质粒pSBPTQ在枯草杆菌DB1342中仍旧保持在83%以上。

Construction of an inducible and efficient expression-secretion shuttle vector of B. subtilis

A new shuttle vector pSB which could replicate in both Escherichia coli and Bacillus subtilis was constructed by fusing the E. coli plasmid pSP72 with the B. subtilis plasmid pUB18. After the sacB promoter and signal peptide sequence of B. subtilis was inserted in the multiple cloning sites (MCS) of pSB, The recombinant plasmid was designated as pSBP. The alpha-amylase gene terminator of Bacillus licheniformis was inserted at the other end of the MCS, resulting in expression-secretion plasmid pSBPT. After a positive control gene degQ of Bacillus pumilus was then cloned into pSBPT, and the inducible and efficient expression-secretion shuttle vector pSBPTQ was thus constructed. To identify the function and the necessary of sacB p. s., alpha-amylase terminator, and degQ in the expression of heterologous gene of pSBPT, the DNA fragment encoding for vasostatin I was cloned downstream of sacB p. s. of pSBP, pSBPT and pSBPTQ, and the resultant plasmid pSV, pSVT and pSVTQ were then transformed into B. subtilis strain DB1342. The transformants were screened on LB plates containing 10 microg/mL kanamycin. The positive transformants were separately grown on kanamycin containing 2 x MSR medium and sucrose was added to 2% final concentration for induction after 2h cultivation. The culture supernatant was used to run SDS-PAGE and Western blot. The results show that after induced by sucrose, very few recombinant Vasostatin I was expressed in DB1342(pSV), and recombinant Vasostatin I expressed in DB1342(pSVTQ) with the positive control gene degQ is more than in DB1342(pSVT) without degQ, suggesting that the Vasostatin I gene expression in pSVTQ was enhanced by degQ. Comparing the recombinant Vasostatin I in DB1342(pSVTQ) cells and its culture supernatant, the SDS-PAGE result show that most of the recombinant Vasostatin I was secreted into the culture supernatant and there is no Vasostatin in inclusion body, the secretion rate is about 90%. The result of plasmid stability test show that pSBPTQ maintains at 83% in B. subtilis after 40 generations.