家蝇抗菌肽AMPs17蛋白原核表达条件的优化及其抗真菌活性检测
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摘要
目的:优化AMPs17重组蛋白的原核表达条件,分析重组蛋白的抗真菌活性。方法:比较不同的诱导温度(25℃、28℃、30℃、32℃、34℃)、异丙基硫代-β-D半乳糖苷(IPTG)诱导浓度(0. 025mmol/L、0. 05mmol/L、0. 1mmol/L、0. 3mmol/L、0. 5mmol/L、0. 8mmol/L、1. 0mmol/L)和诱导时间(12h、15h、18h、21h、24h)对AMPs17重组蛋白表达量的影响,筛选AMPs17重组蛋白的最佳表达条件;采用镍离子金属螯合剂亲和层析柱对重组蛋白进行纯化,SDS-PAGE和ImageJ图像分析系统对表达结果进行分析,Western blot对AMPs17重组蛋白进行鉴定,高效液相色谱分析重组蛋白的纯度,微量液体稀释法及菌落计数法检测其抗真菌活性。结果:在诱导温度为32℃、IPTG浓度为0. 05mmol/L的条件下诱导培养15h,AMPs17重组蛋白的表达量最高且最为稳定;HPLC色谱仪分析显示AMPs17重组蛋白纯度可达到90%以上;优化后的AMPs17重组蛋白能有效抑制白色念珠菌的生长。结论:优化了家蝇抗菌肽AMPs17的诱导表达条件,获得了高表达、稳定且具有抗真菌活性的蛋白质,为后续抗菌机制及应用研究提供一定的实验基础。
Objective: To optimize the prokaryotic expression conditions of AMPs17 recombinant protein and analyze the antifungal activity of recombinant protein. Methods: Compare different induction temperatures (25℃,28℃,30℃,32℃,34℃),isopropylthio-β-D galactoside (IPTG) induced concentration (0. 025 mmol/L,0. 05 mmol/L,0. 1 mmol/L,0. 3 mmol/L,0. 5 mmol/L,0. 8 mmol/L,1. 0 mmol/L) and induction time (12 h,15 h,18 h,21 h,24 h) on the expression of AMPs17 recombinant protein,screening the optimal expression conditions of AMPs17 recombinant protein. The recombinant protein was purified by nickel ion metal chelator affinity chromatography column,and the expression results were analyzed by SDS-PAGE electrophoresis and ImageJ image analysis system. The recombinant protein of AMPs17 was identified by Western blot and the purity of the recombinant protein was analyzed by high performance liquid chromatography (HPLC). The antifungal activity was detected by a micro liquid dilution method and a colony counting method. Results: The results showed that the expression of AMPs17 recombinant protein was the highest and the most stable when induced at32℃ and IPTG concentration of 0. 05 mmol/L for 15 h. The HPLC analysis showed that the purity of AMPs17 recombinant protein reached 90%. In addition,AMPs17 recombinant protein can effectively inhibit the growth of Candida albicans. Conclusion: The induction and expression conditions of antibacterial peptide AMPs17 were optimized,and proteins with high expression,stability and antifungal activity were obtained,which provided certain experimental basis for the follow-up antibacterial mechanism and application research.
Objective: To optimize the prokaryotic expression conditions of AMPs17 recombinant protein and analyze the antifungal activity of recombinant protein. Methods: Compare different induction temperatures (25℃,28℃,30℃,32℃,34℃),isopropylthio-β-D galactoside (IPTG) induced concentration (0. 025 mmol/L,0. 05 mmol/L,0. 1 mmol/L,0. 3 mmol/L,0. 5 mmol/L,0. 8 mmol/L,1. 0 mmol/L) and induction time (12 h,15 h,18 h,21 h,24 h) on the expression of AMPs17 recombinant protein,screening the optimal expression conditions of AMPs17 recombinant protein. The recombinant protein was purified by nickel ion metal chelator affinity chromatography column,and the expression results were analyzed by SDS-PAGE electrophoresis and ImageJ image analysis system. The recombinant protein of AMPs17 was identified by Western blot and the purity of the recombinant protein was analyzed by high performance liquid chromatography (HPLC). The antifungal activity was detected by a micro liquid dilution method and a colony counting method. Results: The results showed that the expression of AMPs17 recombinant protein was the highest and the most stable when induced at32℃ and IPTG concentration of 0. 05 mmol/L for 15 h. The HPLC analysis showed that the purity of AMPs17 recombinant protein reached 90%. In addition,AMPs17 recombinant protein can effectively inhibit the growth of Candida albicans. Conclusion: The induction and expression conditions of antibacterial peptide AMPs17 were optimized,and proteins with high expression,stability and antifungal activity were obtained,which provided certain experimental basis for the follow-up antibacterial mechanism and application research.
引文
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[23]彭传林.家蝇抗真菌肽MAF-1原核表达体系优化与活性分析.贵阳:贵阳医学院,2015.Peng C L.Optimization and activity analysis of prokaryotic expression system of Musca domestica antifungal peptide MAF-1.Guiyang:Guiyang Medical College,2015.
[2]Bernard E,Kreis B,Lotte A,et al.Tuberculosis in pregnancy.Clinics in Perinatology,2016,4(2):880.
[3]李秀云.盐酸氨溴索与氟康唑联合对抗耐药白色念珠菌的作用及机制研究.济南:山东大学,2017.Li X Y.The effect and mechanism of ambroxol hydrochloride combined with fluconazole against Candida albicans.Jinan:Shandong University,2017.
[4]Yeaman M R,Büttner S,Thevissen K.Regulated cell death as a therapeutic target for novel antifungal peptides and biologics.[2018-06-28].https://www.ncbi.nlm.nih.gov/pubmed/29854086/.
[5]Wang G,Li X,Wang Z.APD3:the antimicrobial peptide database as a tool for research and education.Nucleic Acids Research,2015,44(D1):1087-1093.
[6]Kounatidis I,Ligoxygakis P.Drosophila as a model system to unravel the layers of innate immunity to infection.Open Biology,2012,2(5):120075.
[7]Panteleev P V,Balandin S V,Ivanov V T,et al.A therapeutic potential of animal I2-hairpin antimicrobial peptides.Current Medicinal Chemistry,2017,24(17):1724-1746.
[8]Park J,Kang H K,Choi M C,et al.Antibacterial activity and mechanism of action of analogues derived from the antimicrobial peptide m BjAMP1 isolated from Branchiostoma japonicum.Journal of Antimicrobial Chemotherapy,2018,73(8):2059.
[9]Lyu Y,Yang Y,Lyu X,et al.Antimicrobial activity,improved cell selectivity and mode of action of short PMAP-36-derived peptides against bacteria and Candida.Scientific Reports,2016,6(2):27258.
[10]Zhou J,Kong L,Fang N,et al.Synthesis and functional characterization of MAF-1A peptide derived from the larvae of housefly,Musca domestica(Diptera:Muscidae).Journal of Medical Entomology,2016,53(6):1467-1472.
[11]Velden W J V D,Iersel T M V,Blijlevens N M,et al.Safety and tolerability of the antimicrobial peptide human lactoferrin 1-11(h LF1-11).BMC Medicine,2009,7(1):44.
[12]Mehta S,Singh C,Plata K B,et al.Beta-lactams increase the antibacterial activity of daptomycin against clinical methicillinresistant Staphylococcus aureus strains and prevent selection of daptomycin-resistant derivativ.Antimicrobial Agents and Chemotherapy.2012,56(12):6192-6200.
[13]Craik D J,Fairlie D P,Liras S,et al.The future of peptidebased drugs.Chemical Biology and Drug Design,2013,81(1):136-147.
[14]Matejuk A,Begum Q,Woodle M C.Peptide-based antifungal therapies against emerging infections.Drugs Future,2010,35(3):197-231.
[15]Scott J G,Liu N,Kristensen M,et al.A case for sequencing the genomeof Musca domestica(Diptera:Muscidae).Journal of Medical Entomology,2009,46(2):175-182.
[16]Ai H,Wang F,Zhang N,et al.Antiviral,immunomodulatory,and freeradical scavenging activities of a protein-enriched fraction from the larvae of the housefly,Musca domestica.Journal of Insect Science,2013,13(112):112
[17]陶如玉,李妍,马慧玲,等.家蝇AMP17基因的克隆及其分子特性和表达模式研究.中国病原生物学杂志,2017,12(11):1079-1083.Tao R Y,Li Y,Ma H L,et al.Cloning and molecular characteristics and expression patterns of AMP17 gene in Musca domestica.Chinese Journal of Pathogenic Biology,2017,12(11):1079-1083.
[18]王中原.可溶性人SUMO融合表达系统的研究.长春:吉林大学,2010.Wang Z Y.Study on soluble human SUMO fusion expression system.Changchun:Jilin University,2010.
[19]Nuc P,Nuc K.Recombinant protein production in Escherichia coli.Postepy Biochem,2006,52(4):448-456.
[20]Gao Q,Zhao J M,Song L S,et al.Molecular cloning,characterization and expression ofheat shock protein 90 gene in the haemocytes of bay scallop Argopecten irradiants.Fish&Shellfish Immunology,2008,24(4):379-385.
[21]Liang R,Liu X,Liu J,et al.A T7-expression system under temperature control could create temperature-sensitive phenotype of target gene in Escherichia coli.Journal of Microbiological Methods,2007,68(3):497-506.
[22]解庭波.大肠杆菌表达系统的研究进展.长江大学学报(自科版),2008,5(3):77-82.Xie T B.Research progress of E.coli expression system.Journal of Yangtze University(Self version),2008,5(3):77-82.
[23]彭传林.家蝇抗真菌肽MAF-1原核表达体系优化与活性分析.贵阳:贵阳医学院,2015.Peng C L.Optimization and activity analysis of prokaryotic expression system of Musca domestica antifungal peptide MAF-1.Guiyang:Guiyang Medical College,2015.