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英文摘要
For the demands of biological research and industrial purpose, the T7 RNA polymerase (T7 RNAP)/T7 promoter specific expression system has been established in Escherichia coli and other hosts. However, certain limitations of these systems do exist such as the stability of microorganism at high temperature, and the mechanism of secretion etc. Because the physiological and genetical characteristics of B. subtilis are clear, the aim of this project is trying to construct a constitutive T7 expression system without inducer in B. subtilis. In B. subtilis, P1P2 promoter is a part of the rpoD operon which encodes for ORF23, DnaG and SigA (s43). The P1P2 promoter actively expresses in the vegetative cells. Plasmid pHP13, a shuttle vector of E. coli and B. subtilis, was used for the construction of T7 RNAP gene under P1P2 promoter control. These constructions were confirmed by restriction enzyme analysis and DNA sequencing and designed as plasmids pMW190 and pMW221 with various sources of T7 RNAP fragment, respectively. Plasmids pMW190 or pMW221 were transformed into a protease deficient strain B. subtilis DB430 and coexisted with pMW230 (a reporter plasmid under T7 promoter control). No target protein was detected in these B. subtilis strains by Western blot analysis. T7 RNAP activity were determinated by Slot blotting, a much higher transcription activity was detected in B. subtilis than in E. coli. The results indicate that the higher expression of T7 RNAP in B. subtilis may have an inhibitory effect on the translation process. Therefore, a single copy of P1P2-T7 RNA gene was constructed using the transcriptional fusion vector pDH32. The resultant plasmid was integrated into the chromosome by homologous recombination, and the transcriptional activity of P1P2 promoter was analysed by measuring b-galactosidase activity at vegetative and sporulation phases. However, no target protein expression was detected. In vitro transcription assay indicated that both pMW190 and pMW221 can express a functional T7 RNAP protein, and both are capable of turning on the transcription of T7 promoter in vitro. The above results indicate that T7 RNAP under P1P2 promoter control has transcriptional and translational activity. The reason why T7 promoter can not be recognized by a functional T7 RNAP in B. subtilis system requires further study.
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