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Objective: Identification of the attachment site of phage PaP3 within the genome of Pseudo-monas aeruginosa PAS. Methods:The full genome of lysogenic bacteria was cleaved by Pst Ⅰ and produce a large fragment of more than 45 000 bp, which was subsequently digested by EcoR Ⅰ. Then the fragment containing DNA sequence of phage and bacteria was cloned into pFastBacTMHT A vector, and the result of sequencing indicated the right hybrid site attR. AttL was isolated by PCR on the base of integration mechanism. And then attP and attB were indentified according to the nucleotide sequences of attR and attB. Results:A sequence of 21 bp(5'-GGTCGTAGGTTCGAATCCTAC-3') was defined to be the core site of integration, which was located at t-RNAPro gene in the genome of phage PaP3 and t-RNALys gene in the genome of Pseudomonas aeruginosa PA3. The attP and attB flanked with a set of inverted repeat and direct repeat. Conclusion:The integrated site of PaP3 within the genome of PA3 was identified and characteriged, which could be of value in investigating the mechanism of integration and gene flow between different species in the natural world.
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Objective: To screen the best genetic engineering bacterium for the production of peptide antibiotic hPAB-? and evaluate its fermentation level in bottle. Methods:After analysis of the interest fusion protein expression levels of 8 recombinant bacteria containing 1-8 copies of human peptide antibiotic hPAB-? expressing plasmid respectively,2-5 copies expressing bacteria were chosen for the further study of their bacteria yield,expression forms of the target protein, dissolution of the inclusion bodies and the efficiency of fusion protein purification by affinity chromatography, then the best engineering bacterium with the certain copies of interest peptide expressing plasmid was screened out and its optimal fermentation parameters in bottle were also studied. Results:The recombinant bacterium transformed by 3 copies of interest peptide expressing plasmid was the best candidate for its bacteria yield (3.153 g/L) and fusion protein expression level (27.7%) were the highest among 1-8 copies candidates. The inclusion bodies of 3 copies target fusion protein could be easily dissolved by 8 mol/L urea and captured by Ni-NTA column. The elution of the fusion protein could be directly cleaved to monomer by adding 2 mol/L hydroxylamine, adjusting pH to 9.0 and incubating at 45℃ for 2 h. The optimal fermentation conditions of the selected recombinant bacteria were: culture the organisms with modified M9-CAA media at 37℃ and 160 r/min to (A 600 )≈2.5, then add IPTG to the final concentration 100 ?mol/L to induce the expression of target fusion protein for 5 h. Conclusion:The engineering bacterium containing 3 copies interest peptide recombinant expressing plasmid is the best candidate for the production of peptide antibiotic hPAB-?,and its fermentation parameters are confirmed.
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Objective To study the effectrve approach of the nutrition support in burn patients. Methods The means of immuno-precipitation-deduction,ELISA and fluore-photometion were used to test the change of activities,protein expression of the 19S regulator and the rate of protein degradation in skeletal muscle in scard rats with enteral feeding or parenteral nutrition. Results Compared with parenteral nutrition , enteral feeding could markedly reduce the activity and protein expression of the 19S regulator ,and the digeneration of skeletal muscle was also lower. Conclusions The early enteral feeding can distinctly inhibit the system of 26S proteasome , thereby reduce the protein degradation of skeletal muscle in scald rats,which may be benefical to the metabolic modulation of the burned patients.
ABSTRACT
The ubiquitin-dependent 20s/26s proteasome system is the capital pathway of exo-lysosome proteolysis within eukaryotic cell. Under conditions of denervation, starvation, glucocorticoid, infection, tumor, burn and so on, the proteasome system was stimulated to degrade protein, which results in muscle lose fast. Glucocorticoid, insulin, thyroid hormone,TNF? and IL-1? play important roles in the regulation of muscle protein degradation and the proteasome system. Inhibition or activation of the proteasome system was approved to be a novel means of treatment with cachexia and negative nitrogen balance. [
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Objectives:To compare the effect of defferent routes of nutritional support on the 26 S proteasome. Methods:The means of immuno precipitation deduction,ELISA and fluore photomction were used to test the change of activities,contents of the 26 S proteasome and the rate of protein degradation in skeletal muscle of scalding rats with the animal model of enteral feeding and parenteral nutrition. Results:Compared with parenteral nutrition,enteral feeding could markedly reduce the activity and content of the 26 S proteasome,and the release of tyrosine was also lowered. Conclusions:The early enteral feeding can distinctly inhibit the system of 26 S proteasome,and reduce the protein degradation in skeletal muscle in bruned rats.
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Objective To explore the mechanism of negative nitrogen balance in the treatment after burns. Methods The means of immuno precipitation deduction and ELISA were used to test the activities and contents of 26S proteasome and 19S regulator in skeletal muscle of rats inflicted with 30%TBSAⅢ burns. Results TNF? markedly raised the activities and contents of 26S proteasome and 19S regulator in skeletal muscle after scalding. Conclusion TNF? activates the 26S proteasome system in skeletal muscle, thus it enhances the degradation of protein, which is associated with the development of negative nitrogen balance following scalding.
ABSTRACT
To elucidate the mechanism of negative nitrogen balance after burns, immuno precipitation deduction was used to observe the changes in the activity of 26S proteasome and 19S regulator in skeletal muscle of rats inflicted with 30%TBSAⅢ o burns. The protease activity, ATPase activity of 26S proteasome,as well as 19S regulator in skeletal muscle of rats were markedly enhanced on the second day after scald. It is suggested that burn injury could activate the 26S proteasome in skeletal muscle, in turn enhance the degradation of protein , which is associated with negative nitrogen balance following scald