转基因水稻中重组人乳铁蛋白的分离纯化及其抑菌活性研究

目的研究从转基因水稻中分离纯化重组人乳铁蛋白(recombinant humanlactoferrin,rhLF),并检测其是否具有抑菌活性。方法通过饱和硫酸铵分级沉淀从转基因水稻中分离rhLF后,经CM Sepharose FF交换层析和Sephadex G25分子筛层析进一步纯化rhLF。采用微量肉汤稀释法检测不同浓度rhLF(0.25、0.50、1.00、2.00、4.00和5.00mg/ml)对鼠伤寒沙门菌、金黄色葡萄球菌、蜡样芽孢杆菌、单增李斯特菌的抑制情况。结果通过分离和纯化成功获得纯度较高的rhLF(纯度约90%),经考马斯亮蓝染色,Western blot和质谱仪鉴定分析确定为rhLF,分子量约为79 kD。抑菌实验显示,5.00和4.00mg/ml rhLF对鼠伤寒沙门菌和金黄色葡萄球菌有持久的抑制效果,2.00和1.00mg/ml rhLF在实验后期也表现出明显的抑菌作用,0.50mg/ml及以下浓度则没有抑菌效果。5.00和4.00mg/ml rhLF在18h内对蜡样芽孢菌表现出一定的抑制效果;5.00mg/ml rhLF对单增李斯特菌在18h内有抑制作用。结论从转基因水稻中可成功分离和纯化rhLF,纯化后蛋白仍具有明显的抑菌活性。

Isolation and purification of recombinant human lactoferrin (rhLF)from transgenic rice and its antibacterial activities

Objective To study the isolation and purification of recombinant human lactoferrin(rhLF) from transgenic rice,and to check its antibacterial activities.MethodsAfter isolatated rhLF from transgenic rice via saturated ammonium sulfate precipitation,then purified it through CM Sepharose FF-exchange chromatography and molecular sieve chromatography Sephadex G25.The inhibition effects under different concentrations of rhLF(0.25,0.5,1.0,2.0,4.0,and 5.0mg/ml) against Salmonella typhimurium,Staphyloccocus aureus,Bacillus cereus,Listeria monocytogenes were observed,using broth microdilution method.Results The rhLF was obtained at a higher purity(about 90%) through successful isolation and purification.After Coomassie blue staining,Westernblot and mass spectrometer analysis,it was identified as the purpose protein with the molecular weight of approximately 79 kDa.The antibacterial experiments showed that 5mg/ml and 4mg/ml rhLF could inhibite Salmonella typhimurium and Staphylococcus aureus persistently,2mg/ml and 1mg/ml rhLF showed a significant inhibitory effects in the later period;while 0.5mg/ml or lower concentration,showed no inhibitory effects.As to Bacillus cereus,only 5mg/ml and 4mg/ml rhLF exhibited certain inhibitory effects within 18 hours.Listeria monocytogenes was inhibited within 18 hours just at 5mg/ml rhLF.Conclusion The rhLF could be successfully separated and purified from transgenic rice,and the purified protein still has significant antibacterial activities.