猪乳铁蛋白在大肠杆菌中的表达及其活性研究  

Study on porcine lactoferrin expressing in E.coli and activity

作　　者：唐丽杰[1,2] 黄薇薇[1] 于慧[1] 葛俊伟[2] 乔薪瑗[2] 李一经[2] 

机构地区：[1]东北农业大学大学生命科学学院,哈尔滨150030 [2]东北农业大学动医学院,哈尔滨150030

出　　处：《东北农业大学学报》2013年第12期16-21,共6页Journal of Northeast Agricultural University

基　　金：国家自然科学基金(31072057;30871809);黑龙江省教育厅创新团队基金(2011TD001)

摘　　要：乳铁蛋白(Lactoferrin,LF)是一种天然活性铁离子结合糖蛋白,具有抗菌、抗病毒及调节免疫系统等功能。本研究将全基因合成的猪乳铁蛋白基因(Porcine Lactoferrin,PLF)在大肠杆菌(Escherichia coli)BL21(DE3)中进行表达,表达产物主要以包涵体的形式存在;通过对温度、时间及诱导剂浓度等表达条件的摸索提高其表达量;表达的包涵体经裂解和纯化后,对金黄色葡萄球菌(Staphylococcus aureas)以琼脂扩散法进行体外抑菌活性检测,并以透射电镜观察法研究猪乳铁蛋白对该菌的抑制作用机制;检测重组猪乳铁蛋白体外对水疱性口炎病毒(VSV)在MDCK(Madin-Daby canine kidney)细胞上的增殖抑制作用。结果表明,PLF在大肠杆菌中成功表达约79ku;重组菌在32℃经0.8 mmol·L-1IPTG诱导3 h可获得最佳表达量,重组蛋白占菌体蛋白的31.4%;琼脂孔穴扩散抑菌圈法结果表明0.1 mg的重组猪乳铁蛋白对金黄色葡萄球菌的最大抑菌直径达15 mm,透射电镜观察可见经重组猪乳铁蛋白作用后,细菌细胞壁和胞浆内电子密度都降低,菌体细胞壁及细胞膜出现断裂,核心溶解,菌体近似空壳;体外抗病毒试验结果表明,该蛋白可以明显的抑制VSV在MDCK细胞上细胞病变的产生。结果显示,重组的猪乳铁蛋白具有一定的抗菌和抗病毒活性,为其在生产中进一步应用奠定基础。Lactoferrin （ Lactoferrin, LF ） is a kind of natural active iron binding glycoprotein, has functions of antibacterial, antiviral and adjust immune system etc. In this study, the gene synthetic of porcine lactoferrin （PLF） gene was expressed in E. coil BL21 （DE3）, the expressed products mainly existed in the form of inclusion body; in order to improve the amount of the recombinant protein, we explored the expression conditions of temperature, time and inducer concentration; after the extraction and purification of the expressed inclusion bodies, the recombinant PLF protein was carried out in the agar diffusion method in vitro antimicrobial activity against Staphylococcus aureus, and transmission electron microscopy method to explore the mechanism of the recombinant PLF protein＇s inhibition to Staphylococcus aureus; detection effect of the recombinant PLF protein in vitro inhibition of vesivular stomatitisvirus （VSV） proliferation on MDCK cells. The results showed that, approximately 79 ku recombinant porcine lactoferrin had been expressed in the recombinant strain pET-30b-PLF/BL21 （DE3） by SDS-PAGE and Western-blot detection, its consistent with the theoretical value of porcine lactoferrin. The ultrosonic results showed that the recombinant protein existed as inclusion body, the optical expression conditions of the recombinant strain was that it was induced by 0.8 mmoI.L1 IPTG and cultured at 32 ~C and for 3 h then the recombinant protein would accounted for 31.4% of the total bacterial protein; agar diffusion method in vitro antibacterial test results showed that the maximum inhibition diameter of recombinant porcine lactoferrin on Staphylococcus aureus was 15 mm, transmission electron microscopy observation shows that by the role of recombinant porcine lactoferrin, the electron density in the bacterial cell wall and cytoplasm are lower, the bacterial cell wall and cell membrane breaking, the core dissolved cell approximation of shell; antiviral experimental results show that PLF could inhib

关 键 词：猪乳铁蛋白 大肠杆菌表达 抑菌活性 抗病毒活性 

分 类 号：Q93[生物学—微生物学]