[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.