Tandem expression and anti-Candida albicans activity determination of antimicrobial peptide MAF-1A from Musca domestica
MA Xiao-lin1, 2, ZHU Zhi-cui1, 2, ZHANG Ying-chun1, WU Jian-wei1, WANG Tao1, 2
1. School of Basic Medical Science, Guizhou Medical University, Guiyang 550025, China; 2. Key Laboratory of Medical Microbiology and Parasitology of Guizhou Province, Guiyang 550025, China
Abstract:We constructed the prokaryotic expression system of antimicrobial peptide MAF-1A gene, expressing MAF-1A with antifungal activity in Escherichia coli. According to E.coli codon usage, we optimized cDNA sequence of MAF-1A. After that, the 5 multimeric MAF-1A genes were synthesized and then cloned into prokaryotic expression vector pET-28a. The combinant vector was transformed into E. coli Roseeta(DE3) and inducted with IPTG. The production was analyzed with SDS-PAGE electrophoresis and Western Blot. The recombinant 5×MAF-1A protein was purified by use of Ni-NTA and cleaved specially by Enterokinase to generate the MAF-1A monomer. Antifungal activity of MAF-1A monomer against Candida albicans was determined with microdilution method. The 5×MAF-1A protein was successfully expressed in E. coli with solubility and the maximum expression was 2.0 mg/mL. The MIC and MBC of MAF-1A against C.albicans were 0.5 mg/mL and 1.0 mg/mL respectively. The prokaryotic expression system of MAF-1A was constructed successful and could be expressed efficiently in E. coli cells with higher C. albicans activity.
马晓琳,朱志翠,张迎春,吴建伟,王涛. 家蝇抗菌肽MAF-1A的串联表达与抗白念珠菌体外活性检测[J]. 中国人兽共患病学报, 2018, 34(5): 429-433.
MA Xiao-lin, ZHU Zhi-cui, ZHANG Ying-chun, WU Jian-wei, WANG Tao. Tandem expression and anti-Candida albicans activity determination of antimicrobial peptide MAF-1A from Musca domestica. Chinese Journal of Zoonoses, 2018, 34(5): 429-433.
[1] Lo HJ, Tseng KY, Kao YY, et al.Cph1p negatively regulates MDR1 involved in drug resistance in Candida albicans[J]. Int J Antimicrob Agents, 2015, 45(6): 617-621. DOI: 10.1016/ j. ijantimicag. 2015. 01. 017 [2] Guilhelmelli F, Vilela N, Smidt KS, et al.Activity of scorpion venom-derived antifungal peptides against planktonic cells of Candida spp. and Cryptococcus neoformans and Candida albicans biofilms[J]. Front Microbiol, 2016, 7(B4698): 1844. DOI: 10. 3389/ fmicb. 2016. 01844 [3] Pfaller MA.Antifungal drug resistance: mechanisms, epidemiology, and consequences for treatment[J]. Am J Med, 2012, 125(1 Suppl): S3-13. DOI: 10.1016/j. amjmed. 2011. 11. 001 [4] Wang G, Mishra B, Lau K, et al.Antimicrobial peptides in 2014[J]. Pharmaceuticals, 2015, 8(1): 123-150. DOI: 10. 3390/ph8010123 [5] Zhan YO, Wiradharma N, Yi YY.Strategies employed in the design and optimization of synthetic antimicrobial peptide amphiphiles with enhanced therapeutic potentials[J]. Adv Drug Delivery Rev, 2014, 78: 28-45. DOI: 10. 1016/j. addr. 2014. 10.013 [6] Kocourková L, Novotná P, Cujová S, et al.Conformational study of melectin and antapin antimicrobial peptides in model membrane environments[J]. Spectrochim Acta A MolBiomol Spectrosc, 2017, 170: 247-255. DOI: 10. 1016/j. saa. 2016. 07. 015 [7] Wang T, Xiu J, Zhang Y, et al.Transcriptional responses of Candida albicans to antimicrobial peptide MAF-1A[J]. Front Microbiol, 2017, 8: 894. DOI: 10.3389 / fmicb. 2017. 00894 [8] 罗振华,吴建伟,付萍,等.人工合成家蝇抗真菌肽MAF-1A对白色念珠菌致病性的影响[J].第三军医大学学报, 2013, 35(20): 2203-2207. [9] Kusmiati K, Priadi D, Rahayu RKB.Antibacterial activity test, evaluation of pharmacognosy and phytochemical screening of some extracts of globe amaranth (gomphrena globosa)[J]. J Pure Appl Chem Res, 2017, 6(1): 27-33. DOI: 10.21776/ub. jpacr. 2017. 006. 01. 288 [10] John HR, Borbara DA, David A, et al.Reference method for broth dilution antifungal susceptibility testing of yeasts; Approved standard-Third Edition[M]. Clinical and Laboratory Standards Institute document M27-A3, 2008, 28(14): 1-40 [11] Klint JK, Senff S, Saez NJ, et al.Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli[J]. PLoS One. 2013, 8(5): e63865. DOI: 10.1371/ journal. pone. 0063865 [12] 祁丽,姜宁,张爱忠,等. 抗菌肽研发现状及其改造策略[J].中国畜牧兽医,2016, 43(2): 450-456.DOI:10.16431/j.cnki.1671-7236.2016.02.024 [13] Yi T, Sun S, Huang Y, et al.Prokaryotic expression and mechanism of action of helical antimicrobial peptide A20L using fusion tags[J]. Bmc Biotechnol, 2014, 15(1): 1-10. DOI: 10. 1186/s12896-015-0189-x [14] 贾红,袁维峰,李杰,等. 细粒棘球蚴EG95s重组蛋白串联表达免疫原性分析[J]. 中国人兽共患病学报, 2014, 30(8): 843-847.DOI:10.3969/j.issn.1002-2694.2014.08.014 [15] 易方浩,张俊爱,黎四平,等. 4种血清型登革病毒多表位重组抗原的表达及血清学初步评价[J]. 中国人兽共患病学报,2017,33(1): 32-37.DOI:10.3969/j.issn.1002-2694.2017.01.006 [16] 郑华,杨柳,付利芝,等.串联线性抗菌肽在大肠杆菌中的表达及活性抗菌肽的制备[J]. 黑龙江畜牧兽医, 2015,9: 192-194.DOI:10.13881/j.cnki.hljxmsy.2015.0711 [17] Saugandhika S, Sharma V, Malik H, et al.Molecular characterization of IFN-T expressed in buffalo embryonic trophoblasts and expression of recombinant BuIFN-T1a2 and BuIFN-T8 isoforms in E. coli[J]. Protein Exp Purificat, 2016, 122: 8-14. DOI: 10. 1016/j. pep. 2016. 02. 005 [18] 成娟丽,李锋,王红,等.抗菌肽MagaininⅡ多拷贝串联体的设计与构建[J].生物技术, 2014, 24(4): 75-79. [19] Xu Y, Wang Q, Hang B, et al.Serial expression and activity analysis of LNK-16: a bovine antimicrobial peptide analogue[J]. Protein J, 2014, 33(4): 309-31. DOI: 10.1007/ s10930-014-9563-0 [20] Gordon YJ, Romanowski EG, McDermott AM. A review of antimicrobial peptides and their therapeutic potential as anti-infective drugs[J]. Curr Eye Res, 2005, 30(7): 505-515. DOI: 10. 1080/02713680590968637