抗菌肽MAF-1A洐生肽的设计及其抗白色念珠菌活性  

Design of antibacterial peptide MAF-1A derivatives and their activity against Candida albicans

作　　者：黄敏慧 曾晔 张迎春 李彩多 吴建伟 陈峥宏 王涛 HUANG Minhui;ZENG Ye;ZHANG Yingchun;LI Caiduo;WU Jianwei;CHEN Zhenghong;WANG Tao(School of Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China;Key Laboratory of Microbiology and Parasitology of Institution of Higher Learning of Guizhou,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院,贵州贵阳550025 [2]贵州省普通高等学校病原生物学特色重点实验室,贵州贵阳550025 [3]贵州医科大学

出　　处：《贵州医科大学学报》2023年第1期25-32,共8页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81360254);贵州省科技计划项目(黔科合平台人才[2018]5799-22,黔科合支撑[2020]4Y236)。

摘　　要：目的探讨家蝇抗真菌肽-1A(MAF-1A)衍生肽的设计及其抗白色念珠菌(C.albicans)活性。方法采用序列截短及氨基酸残基替换法设计4条MAF-1A衍生肽,生物信息学分析其理化特性;取对数生长期C.albicans ATCC10231制备成浓度为(0.5~2.5)×10^(6)cfu/L的菌液,采用微量稀释法检测4条衍生肽对C.albicans的抗菌活性,另设MAF-1A组(25~1600 mg/L MAF-1A处理)、氟康唑(FLC)对照组(0.25~64.00 mg/L FLC处理);采集健康人全血,离心取血细胞制作重悬红细胞,与不同终浓度(25~400 mg/L)活性衍生肽混匀,分为MAF-1A组、MAF-1A22S3组、MAF-1A20S3组、MAF-1A18S5组、MAF-1A16S5组、阳性对照(0.1%Triton X-l00处理)及阴性对照组[磷酸盐缓冲溶液(PBS)处理],采用体外溶血实验检测各组红细胞的溶血率;制备(1.0~5.0)×10^(9)cfu/L菌液,与不同终浓度[1、2、4×最小抑菌浓度(MIC)]活性衍生肽稀释液混合,设MAF-1A组(1、2、4×MIC MAF-1A处理)和FLC(1、2、4×MIC FLC处理)对照组,分别于0、4、8、12、16、20及24 h取混合菌液100μL于沙氏葡萄糖琼脂培养基(SDA)平板上37℃培养,记录菌落数,绘制时间-杀菌曲线检测MAF-1A、MAF-1A22S3及FLC对C.albicans的杀菌动力学;制备1.0×10^(9)cfu/L C.albicans菌液,加不同终浓度(1、2、4×MIC)的活性衍生肽稀释液,设MAF-1A(1×MIC MAF-1A处理)、FLC(1×MIC FLC处理)对照组和阴性对照组(PBS处理),采用扫描电镜观察各组C.albicans的形态学改变。结果4条衍生肽对C.albicans均具有抗菌活性,MIC和MFC值均较模板肽MAF-1A降低,其中MAF-1A22S3的抗菌活性增加最为明显;衍生肽MAF-1A22S3、MAF-1A20S3溶血活性低于模板MAF-1A,其中MAF-1A22S3的溶血性最低(6×MIC范围内溶血率<5%);MAF-1A22S3对C.albicans具有较强的杀菌作用、且呈浓度依赖性,同浓度时杀菌效率强于模板肽MAF-1A和FLC对照组;衍生肽MAF-1A22S3作用后C.albicans细胞表面出现明显的凹陷、裂痕、胞质外溢、细胞皱缩及裂解死亡等病理改变,�Objective To explore the design of Musca domestica antifungal peptide-1 A(MAF-1 A)and activity against Candida albicans(C.albicans)of its derived peptides.Methods Adopting sequence truncated and amino acid residues substitution to design 4 MAF-1 A derived peptides.Bioinformatics was used to analyze its physical and chemical properties.C.albicans ATCC10231 at logarithmic growth stage was prepared into(0.5-2.5)×10^(6)cfu/L bacterial solution,and the antifungal activity of 4 derived peptides against C.albicans was detected by microdilution method;MAF-1 A group(25-1600 mg/L MAF-1 A treatment)and fluconazole(FLC)group(0.25-64.00 mg/L FLC treatment)were set as control group.Full blood of healthy people was centrifuged and collected to prepare resuspend erythrocyte,and mixed with peptides diluent of different final concentrations(25 to 400 mg/L):MAF-1 A group,MAF-1 A22 S3 group,MAF-1 A20 S3 group,MAF-1 A18 S5 group,MAF-1 A16 S5 group,positive control group(0.1%Triton X-100 treatment),and negative control group[phosphate buffer solution(PBS)treatment].Hemolysis rate of erythrocytes in each group was detected by in vitro hemolysis test.The bacterium suspension of(1.0-5.0)×10^(9) cfu/L was prepared and mixed with active derived peptide diluents of different final concentrations[1,2,4×minimum inhibitory concentration(MIC)];100μL of the mixture was taken on SDA medium at 0,4,8,12,16,20,24 h,cultured at 37℃and record number of colony,respectively;the time-kill curve was drawn to detect the germicidal kinetics of MAF-1 A,MAF-1 A22 S3,and FLC against C.albicans.The solution of C.albicans at concentration of 1.0×10^(9) cfu/L was prepared,and active derived peptide diluents with different final concentrations(1,2,4×MIC)were added;MAF-1 A group(1×MIC MAF-1 A treatment),FLC control group(1×MIC FLC treatment)and negative control group(PBS treatment)were established.Morphological changes of C.albicans in each group were observed by scanning electron microscopy(SEM).Results Four derived peptides had antifungal activities aga

关 键 词：白色念珠菌 抗菌肽 家蝇抗真菌肽-1A 分子改造 衍生肽 抗菌活性 

分 类 号：R379.4[医药卫生—病原生物学]