重组牛乳铁蛋白多肽对沙门菌抑菌作用机制研究  

Antibacterial Mechanism of Recombinant Bovine Lactoferrin Polypeptide on Salmonella

作　　者：李海涛 张宇[2] 刘艳环[2] 桑锐[1] 葛冰洁 周鸿缘 王巍 苗利光[2] 张雪梅[1] LI Hai-tao;ZHANG Yu;LIU Yan-huan;SANG Rui;GE Bing-jie;ZHOU Hong-yuan;WANG Wei;MIAO Li-guang;ZHANG Xue-mei(Agricultural College,Yanbian University,Yanji 133002,China;Institute of Special Wild Economic Animals and Plants,Chinese Academy of Agricultural Sciences,Changchun 130112,China)

机构地区：[1]延边大学农学院,吉林延吉133002 [2]中国农业科学院特产研究所,吉林长春130112

出　　处：《中国兽医杂志》2022年第8期15-20,25,共7页Chinese Journal of Veterinary Medicine

基　　金：中央级公益性科研院所基本科研业务费专项(1610342019011);中国农业科学院科技创新工程(CAAS-ASTIP-2021-ISAPS)。

摘　　要：为探讨重组牛乳铁蛋白多肽(rbLfP)对动物临床常见病原细菌沙门菌的抑菌作用机制,本试验采用微量稀释法测定了rbLfP体外最小抑菌浓度(MIC),并分析了rbLfP对沙门菌细胞膜的损伤作用以及对细菌DNA的相互作用。结果显示,rbLfP对沙门菌体外MIC为32μg/mL,对沙门菌生长具有明显抑制作用;将rbLfP与沙门菌共培养4 h,与空白对照组相比,试验组培养液中的核酸和蛋白质含量明显升高(P<0.05),同时细菌基因组DNA复制明显抑制(P<0.05);rbLfP作用于沙门菌可以增加细菌细胞膜的通透性,导致细胞内容物、金属离子外泄。选用无细胞蛋白质合成系统进行蛋白质合成试验,rbLfP对蛋白质合成的抑制作用呈现一定的剂量依赖效应,8×MIC的rbLfP对蛋白质合成的抑制率近20%,16×MIC的rbLfP抑制效果更明显,说明rbLfP可以通过影响细菌胞内蛋白质的合成来抑杀细菌,且抑杀效果与rbLfP的浓度呈正相关;荧光光谱分析和凝胶阻滞试验结果显示,rbLfP可作用于DNA的磷酸骨架。本试验结果表明,rbLfP可通过改变沙门菌细胞膜的通透性,导致细胞内的核酸和蛋白质外漏,同时其可与细胞内DNA相互作用,抑制菌体DNA复制,起到抑菌作用,并且作用效果与rbLfP的剂量关系呈正相关。本试验揭示了rbLfP对沙门菌抑菌的作用机制,为下一步研究提供了科学依据。In order to uncover the bacteriostatic mechanism of recombinant bovine lactoferrin polypeptide(rbLfP)on Salmonella,a common pathogenic bacterium in animals,the minimum inhibitory concentration(MIC)in vitro was determined by microdilution method,and the damaging effect of rbLfP on Salmonella cell membrane and its interaction with bacterial DNA were studied.The results showed that the MIC of rbLfP against Salmonella in vitro was 32μg/mL,which had significant inhibitory effect on the growth of Salmonella.After rbLfP co-cultured with Salmonella for 4 h,the concentrations of nucleic acid and protein in the culture medium were significantly increased(P<0.05),and the concentration of bacterial genomic DNA in rbLfP group was significantly reduced compared with CK group(P<0.05).By increasing the permeability of cell membrane of Salmonella,rbLfP induced leakage of cell contents and metal ions.Protein synthesis experiments based on a cell-free protein synthesis system revealed a dose-dependent effect of rbLfP on the inhibition of protein synthesis.The inhibition rate of 8×MIC rbLfP on protein synthesis was nearly 20%,and the inhibition effect of 16×MIC rbLfP was more obvious,indicating that rbLfP could inhibit bacteria by affecting intracellular protein synthesis,and the inhibitory effect was positively correlated with the concentrations of rbLfP.The interaction of rbLfP and DNA was analyzed with fluorescence spectrum and gel block test,which proved that rbLfP could interact with the phosphate skeleton of DNA and inhibit the replication of DNA.Thus,rbLfP appears to induce leakage of nucleic acid and protein in Salmonella cells by changing the permeability of cell membrane,and also interacts with DNA in cells to inhibit the replication of DNA,contributing to its antibacterial activity,and the effect is positively correlated with the dose of rbLfP.This experiment revealed the antibacterial mechanism of rbLfP on Salmonella,which provided a scientific basis for further research.

关 键 词：牛乳铁蛋白 抑菌机制 细胞膜损伤 沙门菌 

分 类 号：S855.1[农业科学—临床兽医学]