融合表达HLH结构域对猪溶菌酶抗菌活性的影响  

Effect of fusion expression of HLH domain on antibacterial activity of Sus scrofa lysozyme

作　　者：朱德伟 蔡国林[2] 陆健[2] ZHU Dewei;CAI Guolin;LU Jian(College of Marine and Bio-engineering,Yancheng Teachers University,Yancheng 224051,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China)

机构地区：[1]盐城师范学院海洋与生物工程学院,江苏盐城224051 [2]江南大学工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《生物技术》2024年第1期6-11,90,共7页Biotechnology

基　　金：江苏省自然科学基金青年基金项目(BK20201063)。

摘　　要：[目的]通过遗传操作提高猪溶菌酶抗革兰氏阴性菌的活性。[方法]对猪溶菌酶进行分子模拟,得到了包含功能肽的螺旋-回环-螺旋(HLH)结构域,将HLH编码基因与猪溶菌酶基因N端或C端进行融合,于大肠杆菌中诱导表达,经复性、纯化后检测其抗菌活性,并利用原子力显微镜和荧光染色对抗菌活性较高的融合蛋白杀菌机制进行初探。[结果]与对照组相比,N端和C端融合蛋白基本保持了猪溶菌酶对革兰氏阳性菌的活性;同时,两种融合蛋白对革兰氏阴性菌的抗菌活性均显著增强,其中N端融合产物活性更高,它对大肠杆菌ATCC 10798、大肠杆菌ATCC 25922、克雷伯氏菌TR5、铜绿假单胞菌ATCC 15442、沙门氏菌CMCC(B)50335的抗菌系数分别为1.64、1.24、2.56、1.72和1.42,最低抑菌浓度分别为90μg/mL、100μg/mL、40μg/mL、80μg/mL和100μg/mL。经检测,该融合蛋白能显著增强革兰氏阴性菌细胞膜的通透性。[结论]通过融合表达自身来源的HLH结构域,猪溶菌酶抗革兰氏阴性菌活性得到了显著提升,可为其它溶菌酶抗菌活性的提高提供参考。[Objective]To improve the anti-Gram-negative of Sus scrofa lysozyme(SSL)by genetic manipulation.[Method]A helix-loop-helix(HLH)domain containing a functional peptide was obtained by molecular simulation of SSL.Then,the HLH coding gene was fused with the N-terminal or c-terminal of SSL gene,the recombinant proteins were expressed in E.coli,and their antibacterial activity was detected after renaturation and purification.At last,the bactericidal mechanism of the fusion protein with high antibacterial activity was investigated by atomic force microscopy and fluorescence staining.[Result]Compared with the control group,the N-terminal and c-terminal fusion proteins maintained the activity of SSL against Gram-positive bacteria,and the antibacterial activity of both fusion proteins against Gram-negative was significantly enhanced,the activity ofN-terminal fusion product was higher,the antibacterial coefficients against E.coli ATCC 10798,E.coli ATCC 25922,Klebsiella TR5,Pseudomonas aeruginosa ATCC 15442 and Salmonella CMCC(B)50335 were 1.64,1.24,2.56,1.72 and 1.42 respectively;and the MIC were 90μg/mL,100μg/mL,40μg/mL,80μg/mL and 100μg/mL,respectively.The fusion protein was found to significantly enhance the permeability of the Gram-negative cell membrane.[Conclusion]The anti-Gram-negative of SSL was significantly enhanced by fusion expression of HLH domain,which may provide reference for the activities improvement of other lysozymes.

关 键 词：猪溶菌酶 抗菌活性 抗菌肽 HLH结构域 分子模拟 融合表达 杀菌机制 通透性 

分 类 号：Q812[生物学—生物工程]