| Streptococcus suis is an important zoonotic pathogen that causes meningitis, sepsis, arthritis, endocarditis and pneumonia in piglet or human meningitis. It has become an important disease to seriously affect the pig industry. Meanwhile, it is a big threatens to public hygiene and human life. Worse more, the severe antibiotics resistance of bacteria make it difficult to the prevention and treatment of this disease. In this thesis, Purified active Lysin (LySMP) encoded by phage Lysin gene was prepared by recombinant expression in E. coli, the protocol for preparation and assay of the LySMP was drafted.To produce the purified active LySMP by recombinant expression in E. coli. The DNA of SMP was extracted and a pair of primers was designed according to the sequence of lysin gene. After amplification by PCR, the products were inserted into a prokaryotic expression plasmid, pET-28a(+) to obtain the recombinant plasmid lysin-pET-28a. The recombinant plasmid lysin-pET-28a was then transformed into E. coli BL21(DE3) where it was induced to express protein by IPTG. By changing the temperature of culture, the concentration of IPTG and the time of induction, the expressing conditions for LySMP was optimized. The recombinant protein was purified by Ni-NTA column chromatography and determined by SDS-PAGE as well as Western-blotting. The purified LySMP protein was 5mg/mL.To maximize the biological activity of LySMP in vitro, we analyzed its working conditions. Optimum pH and temperature conditions for the LySMP were investigated by turbidity reduction assay. LySMP exerted efficient activity at 37℃and pH 7.2. The growth curve of 9 Streptococcus suis which preserved and commonly used in the laboratory were determined. By counting the bacteria at logarithmic phase, Mortality rate of 9 Streptococcus suis serotype 2 were statisticed after addition of LySMP within 15 min, respectively. The mortality was 4.6%-36%. LySMP exhibited an extensive lysis spectrum. Salmonella, Listeria, Enterococcus gallinarum, Streptococcus uberis, Streptococcus equi ssp, and Staphylococcus aureus could be lysed and the effect of LySMP lysate is better than purified LySMP. Chromatographically purified LySMP treated with 0.8%β-mercaptoethanol showed no obvious activity increase. Measurement of LySMP activity was based on turbidimetric determination of bacteria lysis, Serial dilutions of LySMP and the reciprocal of the highest dilution that was nearest to half of the control value was defined as the activity of LySMP in unit/mL.LySMP was sensitive to temperature, so it's very important to find the preservation condition of LySMP. Under different temperature conditions, the time keeping the activity of LySMP was different. LySMP will lose activity within 30min at 37℃, while activity could be keep for a week at 4℃, and two or more weeks at -20℃or -80℃. Under -20℃or-80℃freezing conditions, addition of 25% of glycerol preserved most of the LySMP activity. In order to achieve the purpose of long-term preservation of LySMP, the freeze-drying process was investigated. Orthogonal experiment design was taken to choose protective agent for freeze drying.The results from direct calculating and analysis of variance indicated that the protectant system to obtain optimal combinations of levels is 18% sucrose,6% glucose,0.5% glycin,0.8% potassssuim sorbate and 25% glycerol. Using this optimal combination, LySMP activity could maintain 40% after freeze-drying. |
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