银杏抗菌肽蛋白基因原核表达及其重组蛋白的体内外抑菌作用研究(1)
摘 要 目的：獲取银杏抗菌肽(GBA)重组蛋白，并考察其体内外抗菌活性，为解决病原菌耐药问题、规模化生产植物源性新型抗菌剂提供实验基础。方法：根据Genebank中公布的GBA基因序列(FJ865399)，利用基因技术构建重组质粒pET32a(+)-GBA，再以大肠杆菌原核表达获取重组蛋白，并采用凝胶电泳和Western blotting法对所得蛋白进行纯化及鉴定。采用纸片扩散法考察所获重组蛋白对大肠杆菌、金黄色葡萄球菌、绿脓杆菌、鼠伤寒沙门氏菌的药物敏感度;采用肉汤稀释法测定重组蛋白最小抑菌浓度(MIC)和最低杀菌浓度(MBC);考察重组蛋白对金黄色葡萄球菌感染模型小鼠的保护作用。结果：成功表达并纯化获得目标重组蛋白(大小约32 kDa)。该重组蛋白对金黄色葡萄球菌中度敏感，对其他3种细菌低度敏感;对金黄色葡萄球菌的MIC为(50.00±5.00)mg/mL、MBC为(138.33±12.58)mg/mL，MIC显著高于其他3种细菌(P<0.05)。高剂量重组蛋白(8.0 g/kg)能显著降低金黄色葡萄球菌导致的小鼠死亡率(P<0.05)，对小鼠的保护效果与阳性药物青霉素接近。结论：所获重组蛋白对金黄色葡萄球菌的抑制效果明显，对大肠杆菌和绿脓杆菌有一定的抑制作用，对鼠伤寒沙门氏菌抑制作用较弱;该蛋白高剂量给药对金黄色葡萄球菌感染小鼠具有明显的保护作用。
关键词 银杏;抗菌肽;载体质粒;重组蛋白;原核表达;抑菌作用;体内;体外;小鼠
ABSTRACT OBJECTIVE： To obtain Ginkgo biloba antimicrobial peptide (GBA) recombinant protein， and to investigate in vivo/in vitro antimicrobial activity of the protein so as to provide experimental basis for solving bacterial resistance and large-scale production of new plant-derived antimicrobial agents. METHODS： Based on gene technology， according to GBA gene sequence (FJ 865399) published by Genebank， recombinant expression vector plasmid pET32a(+)-GBA was constructed. Prokaryotic expression of recombinant protein was conducted by Escherichia coli， and then the protein was purified and identified by gel electrophoresis and Western blotting. Drug sensitivity of obtained recombinant protein to E. coli， Staphylococcus aureus， Pseudomonas aeruginosa， Salmonella typhimurium were investigated by Kirby-Bauer test. The minimum antibacterial concentration (MIC) and minimum bactericidal concentration (MBC) were determined by broth dilution method. The protective effects of recombinant protein on S. aureus infection model mice were investigated. RESULTS： Target recombinant protein was expressed successfully and purified (molecular weight of 32 kDa). The recombinant protein was moderately sensitive to S. aureus and low sensitive to other three bacterias. MIC and MBC of the recombinant protein to S. aureus were (50.00±5.00)mg/mL and(138.33±12.58)mg/mL， and MIC was significantly higher than those to other 3 kinds of bacterias (P<0.05). High-dose of recombinant protein (8.0 g/kg) could significantly reduce the S. aureus-induced mortality of mice (P<0.05)， and had similar protective effect as positive drug penicillin. CONCLUSIONS： Obtained recombinant protein has obvious antimicrobial effects on S. aureus， inhibits E. coli and P. aeruginosa to certain extent and shows poor inhibitive effect on S. typhimurium. High-dose of recombinant protein shows significant protective effect for S. aureus infection model mice., http://www.100md.com(刘蕾 何光志 王文佳)
关键词 银杏;抗菌肽;载体质粒;重组蛋白;原核表达;抑菌作用;体内;体外;小鼠
ABSTRACT OBJECTIVE： To obtain Ginkgo biloba antimicrobial peptide (GBA) recombinant protein， and to investigate in vivo/in vitro antimicrobial activity of the protein so as to provide experimental basis for solving bacterial resistance and large-scale production of new plant-derived antimicrobial agents. METHODS： Based on gene technology， according to GBA gene sequence (FJ 865399) published by Genebank， recombinant expression vector plasmid pET32a(+)-GBA was constructed. Prokaryotic expression of recombinant protein was conducted by Escherichia coli， and then the protein was purified and identified by gel electrophoresis and Western blotting. Drug sensitivity of obtained recombinant protein to E. coli， Staphylococcus aureus， Pseudomonas aeruginosa， Salmonella typhimurium were investigated by Kirby-Bauer test. The minimum antibacterial concentration (MIC) and minimum bactericidal concentration (MBC) were determined by broth dilution method. The protective effects of recombinant protein on S. aureus infection model mice were investigated. RESULTS： Target recombinant protein was expressed successfully and purified (molecular weight of 32 kDa). The recombinant protein was moderately sensitive to S. aureus and low sensitive to other three bacterias. MIC and MBC of the recombinant protein to S. aureus were (50.00±5.00)mg/mL and(138.33±12.58)mg/mL， and MIC was significantly higher than those to other 3 kinds of bacterias (P<0.05). High-dose of recombinant protein (8.0 g/kg) could significantly reduce the S. aureus-induced mortality of mice (P<0.05)， and had similar protective effect as positive drug penicillin. CONCLUSIONS： Obtained recombinant protein has obvious antimicrobial effects on S. aureus， inhibits E. coli and P. aeruginosa to certain extent and shows poor inhibitive effect on S. typhimurium. High-dose of recombinant protein shows significant protective effect for S. aureus infection model mice., http://www.100md.com(刘蕾 何光志 王文佳)