| Summary: | 碩士 === 國立成功大學 === 微生物暨免疫學研究所 === 87 === For the demands of biological research and industrial purposes, the T7 expression system has been established in Escherichia coli, and other hosts. However, certain limitations of the 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 Bacillus subtilis are known, the aim of this project was trying to construct a spoVA inducible T7 expression system without an inducer in B. subtilis. In B. subtilis, spoVA promoter is controlled by the transcription factor-sigma G. It can express during 3-5 hours after the sporulation initiation. The characters of T7 RNAP are fast of transcription and specific recognition of it’s promoter. Plasmid pHP13, a shuttle vector of E. coli and B. subtilis, was used for the construction of T7 RNAP gene under spoVA promoter control. The construct was confirmed by restriction enzyme analysis and DNA sequencing and designated as pMW205. Plasmid pMW216 contains the truncated streptokinase gene under the T7 promoter control. Both plasmids pMW216 (Emr, copy no.~100 copies/chromosome) and pMW205 (Emr, Cmr) were transformed into DB428, a protease deficient strain. Because of the problem of antibiotic selection, plasmid pMW216 could not co-transform with plasmid pMW205 into strain DB428. In order to solve this problem, pMW230 (Tetr, copy no. ~5 copies/chromosome) was used to construct the expression vector. However, no target protein was detected by western blot analysis. Plasmids pUB110 and pMW209 (E. coli T7 reporter plasmid) were fused for increasing copy number of reporter plasmid and designated as pMW238 (Kanr, copy no.~50 copies/ chromosome),. The target protein could be constitutively expressed without the presence of the plasmid pMW205. These results indicate that reporter plasmid was not controlled by spoVA inducible T7RNAP. Although it could not express target protein by spoVA inducible T7 expression system, the plasmid pUB110, could express small amount of target protein directly.
Aeromonas species are gram-negative bacilli with positive catalase and oxidase reactions. The clinical spectrum is similar to Vibrio vulnificus. It not only causes diseases in aquatic animals, but also in humans. The resistance is easily selected, when cefotaxime (CTX) is treated in vitro. Because the regimen of CTX and minocycline (MIN) combination has been demonstrated to be synergistic against V. vulnificus in vitro and in a murine experiment. MIN is the most active of among three tetracycline analogues. Two clinical isolates of A. hydrophila, A136 and A139, were used to study antibiotics combination against A. hydrophila. Strain A136, cefotaxime-sensitive A. hydrophila, grew rapidly with 6 g/ml of CTX (eight times the MIC) or 6 g/ml of MIN (two times the MIC). But the combination of CTX at 3 g/ml (four times the MIC) and MIN at 2 g/ml (2/3 times the MIC) can inhibit A136 strain at least 48 h. A139, CTX resistant strain also had a similar inhibitory effect with the combination of CTX at 32g/ml and MIN at 4 g/ml (MIC). These results demonstrate that the combination of cefotaxime and minocycline can synergistically against A. hydrophila in vitro. In addition, we also identified an O:11 serogroup and a hybridization group (HG) among Aeromonas clinical isolates. In serogroup O:11 of Aeromonas, the ratio of blood and stool specimens was similar. Therefore, these results no longer support the relationship between serogroup O:11 and invasiveness of Aeromonas infection as was reported previously. The HGs of 75 Aeromonas strains were identified by ribotyping method and the results showed that the distribution of A. hydrophila and A. sobria, but not A. caviae were similar to that of previous report.
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