综述与专题评论

抗菌肽在毕赤酵母中表达的研究进展

  • 查曼 ,
  • 闵勇 ,
  • 刘晓艳 ,
  • 朱镭 ,
  • 邱一敏 ,
  • 陈凌 ,
  • 周荣华 ,
  • 王常高
展开
  • 1(湖北工业大学 生命科学与健康工程学院,湖北 武汉,430068)
    2(湖北省生物农药工程研究中心,湖北 武汉,430064)
    3(湖北省农业科学院植保土肥研究所,湖北 武汉,430064)
第一作者: 硕士研究生(周荣华研究员和王常高副教授为共同通信作者,E-mail:zhouronghua1975@126.com;971963552@qq.com)

收稿日期: 2024-10-09

  修回日期: 2024-11-04

  网络出版日期: 2025-08-04

基金资助

湖北省技术创新计划项目(2024BBB064);湖北省现代农业产业技术体系项目(2023HBSTX4-07);国家自然科学基金项目(32000052)

Research progress on expression of antimicrobial peptides in Pichia pastoris

  • ZHA Man ,
  • MIN Yong ,
  • LIU Xiaoyan ,
  • ZHU Lei ,
  • QIU Yimin ,
  • CHEN Ling ,
  • ZHOU Ronghua ,
  • WANG Changgao
Expand
  • 1(School of Life and Health Science, Hubei University of Technology, Wuhan 430068, China)
    2(Hubei Biopesticide Engineering Research, Wuhan 430064, China)
    3(Institute of Plant Protection and Soil Fertilizer, Hubei Academy of Agricultural Sciences, Wuhan 430064, China)

Received date: 2024-10-09

  Revised date: 2024-11-04

  Online published: 2025-08-04

摘要

抗菌肽是一类由原核生物和真核生物产生的小分子多肽,具有广谱抑菌活性,且不易引发微生物耐药性,因此被认为具有替代抗生素成为新一代抗菌药物的潜力。该文主要综述了抗菌肽的研究进展,并展望了其在医药、畜牧业、农业及食品领域中的应用前景。此外,该文还总结了近年来抗菌肽在毕赤酵母中的重组表达研究,并探讨了提高抗菌肽在该系统中高效表达的优化策略,包括启动子、信号肽、蛋白伴侣的选择、基因剂量及发酵参数的优化。希望通过这些总结,为抗菌肽的应用开发和大规模生产提供有价值的参考。

本文引用格式

查曼 , 闵勇 , 刘晓艳 , 朱镭 , 邱一敏 , 陈凌 , 周荣华 , 王常高 . 抗菌肽在毕赤酵母中表达的研究进展[J]. 食品与发酵工业, 2025 , 51(13) : 354 -361 . DOI: 10.13995/j.cnki.11-1802/ts.041234

Abstract

Antimicrobial peptides (AMPs) are small peptides produced by both prokaryotic and eukaryotic organisms, exhibiting broad-spectrum antibacterial activity and low potential for inducing microbial resistance.As such, AMPs are considered promising candidates to replace antibiotics as the next generation of antimicrobial agents.This paper reviews the recent progress in AMP research and explores their potential applications in medicine, animal husbandry, agriculture, and the food industry.Additionally, it summarizes recent studies on the recombinant expression of AMPs in Pichia pastoris and discusses strategies to enhance their expression efficiency in this system, including the selection of promoters, signal peptides, and molecular chaperones, gene dosage optimization and fermentation parameters.Through these insights, this review aims to provide valuable references for the development and large-scale production of AMPs.

参考文献

[1] AIESH B M, NAZZAL M A, ABDELHAQ A I, et al.Impact of an antibiotic stewardship program on antibiotic utilization, bacterial susceptibilities, and cost of antibiotics[J].Scientific Reports, 2023, 13(1):5040.
[2] COLLABORATORS A R.Global burden of bacterial antimicrobial resistance in 2019:A systematic analysis[J].Lancet, 2022, 399(10325):629-655.
[3] GOW N A R, JOHNSON C, BERMAN J, et al.The importance of antimicrobial resistance in medical mycology[J].Nature Communications, 2022, 13(1):5352.
[4] CHEN L, KUMAR S, WU H Y.A review of current antibiotic resistance and promising antibiotics with novel modes of action to combat antibiotic resistance[J].Archives of Microbiology, 2023, 205(11):356.
[5] MURPHY K E, SLOAN G F, LAWHERN G V, et al.Advances in antibiotic drug discovery:Reducing the barriers for antibiotic development[J].Future Medicinal Chemistry, 2020, 12(22):2067-2087.
[6] RIMA M, RIMA M, FAJLOUN Z, et al.Antimicrobial peptides:A potent alternative to antibiotics[J].Antibiotics, 2021, 10(9):1095.
[7] JI S Q, AN F Y, ZHANG T W, et al.Antimicrobial peptides:An alternative to traditional antibiotics[J].European Journal of Medicinal Chemistry, 2024, 265:116072.
[8] 朱泰承, 李寅.毕赤酵母表达系统发展概况及趋势[J].生物工程学报, 2015, 31(6):929-938.
ZHU T C, LI Y.Recent development of Pichia pastoris system:Current status and future perspective[J].Chinese Journal of Biotechnology, 2015, 31(6):929-938.
[9] SABATIER J M.Antibacterial peptides[J].Antibiotics, 2020, 9(4):142.
[10] BORAHA, DEB B, CHAKRABORTY S.A crosstalk on antimicrobial peptides[J].International Journal of Peptide Research and Therapeutics, 2021, 27(1):229-244.
[11] LI X, ZUO S Y, WANG B, et al.Antimicrobial mechanisms and clinical application prospects of antimicrobial peptides[J].Molecules, 2022, 27(9):2675.
[12] 宋雪莹, 高圣玥, 宋岩, 等.蛙皮抗菌肽及其在畜禽生长性能、免疫功能、消化能力及疾病防治中的作用[J].饲料工业, 2023, 44(11):8-13.
SONG X Y, GAO S Y, SONG Y, et al.Effects of antimicrobial peptides from frog skin on growth performance, immune function, digestive ability and disease prevention of livestock and poultry[J].Feed Industry, 2023, 44(11):8-13.
[13] 卞璐. 昆虫抗菌肽天蚕素的研究进展[J].中国动物保健, 2021, 23(4):116;123.
BIAN L.Research progress of insect antibacterial peptide cecropin[J].China Animal Health, 2021, 23(4):116;123.
[14] CHEN N, JIANG C.Antimicrobial peptides:Structure, mechanism, and modification[J].European Journal of Medicinal Chemistry, 2023, 255:115377.
[15] ZHANG R, XU L J, DONG C M.Antimicrobial peptides:An overview of their structure, function and mechanism of action[J].Protein and Peptide Letters, 2022, 29(8):641-650.
[16] CIULLA M G, GELAIN F.Structure-activity relationships of antibacterial peptides[J].Microbial Biotechnology, 2023, 16(4):757-777.
[17] CHENG K T, WU C L, YIP B S, et al.High level expression and purification of the clinically active antimicrobial peptide P-113 in Escherichia coli[J].Molecules, 2018, 23(4):800.
[18] LIANG X L, YAN J X, LU Y W, et al.The antimicrobial peptide melectin shows both antimicrobial and antitumor activity via membrane interference and DNA binding[J].Drug Design, Development and Therapy, 2021, 15:1261-1273.
[19] DE MOURA G A, DE OLIVEIRA J R, ROCHA Y M, et al.Antitumor and antiparasitic activity of antimicrobial peptides derived from snake venom:A systematic review approach[J].Current Medicinal Chemistry, 2022, 29(32):5358-5368.
[20] MIAO F Z, LI Y, TAI Z G, et al.Antimicrobial peptides:The promising therapeutics for cutaneous wound healing[J].Macromolecular Bioscience, 2021, 21(10):2100103.
[21] YANG M J, TANG M, MA X J, et al.AP-57/C10orf99 is a new type of mutifunctional antimicrobial peptide[J].Biochemical and Biophysical Research Communications, 2015, 457(3):347-352.
[22] LIANG Q P, LIU Z M, LIANG Z Y, et al.Development strategies and application of antimicrobial peptides as future alternatives to in-feed antibiotics[J].Science of The Total Environment, 2024, 927:172150.
[23] ROBLES RAMIREZ O, OSUNA G, PLISSON F, et al.Antimicrobial peptides in livestock:A review with a one health approach[J].Frontiers in Cellular and Infection Microbiology, 2024, 14:1339285.
[24] TAI H M, HUANG H N, TSAI T Y, et al.Dietary supplementation of recombinant antimicrobial peptide Epinephelus lanceolatus piscidin improves growth performance and immune response in Gallus gallus domesticus[J].PLoS One, 2020, 15(3):e0230021.
[25] TOMASINSIG L, DE CONTI G, SKERLAVAJ B, et al.Broad-spectrum activity against bacterial mastitis pathogens and activation of mammary epithelial cells support a protective role of neutrophil cathelicidins in bovine mastitis[J].Infection and Immunity, 2010, 78(4):1781-1788.
[26] SAEED S I, MERGANI A, AKLILU E, et al.Antimicrobial peptides:Bringing solution to the rising threats of antimicrobial resistance in livestock[J].Frontiers in Veterinary Science, 2022, 9:851052.
[27] TANG R, TAN H, DAI Y, et al.Application of antimicrobial peptides in plant protection:Making use of the overlooked merits[J].Frontiers in Plant Science, 2023, 14:1139539.
[28] YU L, YANG M J, JIANG D, et al.Antibacterial peptides from Monochamus alternatus induced oxidative stress and reproductive defects in pine wood nematode through the ERK/MAPK signaling pathway[J].Pesticide Biochemistry and Physiology, 2023, 194:105511.
[29] TIWARI I, BHOJIYA A A, PRASAD R, et al.Putative role of anti-microbial peptide recovered from Lactiplantibacillus spp.in biocontrol activity[J].Current Microbiology, 2024, 81(3):88.
[30] DONG B, LIN Y J, SU Z W, et al.Recombinant human β-defensin130 inhibited the growth of foodborne bacteria through membrane disruption and exerted anti-inflammatory activity[J].Food Science and Biotechnology, 2022, 31(7):893-904.
[31] 朱宏宇, 王晓璐, 刘亚君, 等.产肌醇毕赤酵母细胞工厂的优化[J].微生物学通报, 2023, 50(9):3731-3746.
ZHU H Y, WANG X L, LIU Y J, et al.Optimization of a Komagataella phaffii cell factory for producing inositol[J].Microbiology China, 2023, 50(9):3731-3746.
[32] CEREGHINO J L, CREGG J M.Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J].FEMS Microbiology Reviews, 2000, 24(1):45-66.
[33] LIANG X X, JIANG H, SI X D, et al.Boosting expression level of plectasin in recombinant Pichia pastoris via 2A self-processing peptide assembly[J].Applied Microbiology and Biotechnology, 2022, 106(9-10):3669-3678.
[34] YU D P, ZHAO H H, WEN Y M, et al.Characterization and functional evaluation of NK-lysin from clownfish (Amphiprion ocellaris)[J].Fishes, 2023, 8(11):533.
[35] PIPIYA S O, KUDZHAEV A M, MIRZOEVA N Z, et al.Bioengineering the antimicrobial activity of yeast by recombinant thanatin production[J].Antibiotics, 2023, 12(12):1719.
[36] LI Z X, CHENG Q, GUO H N, et al.Expression of hybrid peptide EF-1 in Pichia pastoris, its purification, and antimicrobial characterization[J].Molecules, 2020, 25(23):5538.
[37] ÇOBANOĞLU Ş, ARSLAN E, YAZıCı A, et al.Expression of human β-defensin 2 (hBD-2) in Pichia pastoris and investigation of its binding efficiency with ACE-2[J].The Protein Journal, 2023, 42(4):399-407.
[38] DONG X F, SHAN H, WANG S B, et al.High expression of antimicrobial peptides cathelicidin-BF in Pichia pastoris and verification of its activity[J].Frontiers in Microbiology, 2023, 14:1153365.
[39] ZHAN N, WANG T Y, ZHANG L C, et al.A eukaryotic expression strategy for producing the novel antimicrobial peptide PRW4[J].Brazilian Journal of Microbiology, 2020, 51(3):999-1008.
[40] DONG C M, LI M R, ZHANG R, et al.The expression of antibacterial peptide turgencin A in Pichia pastoris and an analysis of its antibacterial activity[J].Molecules, 2023, 28(14):5405.
[41] ZHAO L, LI L, HU M Y, et al.Heterologous expression of the novel dimeric antimicrobial peptide LIG in Pichia pastoris[J].Journal of Biotechnology, 2024, 381:19-26.
[42] ZHAO Z H, ZHANG C X, LI J, et al.Effect of tandem repeats of antimicrobial peptide CC34 on production of target proteins and activity of Pichia pastoris[J].Protein Expression and Purification, 2023, 212:106342.
[43] HUO X C, WANG P X, ZHAO F X, et al.High-efficiency expression of a novel antimicrobial peptide I20 with superior bactericidal ability and biocompatibility in Pichia pastoris and its efficiency enhancement to aquaculture[J].Aquaculture, 2024, 579:740149.
[44] HUANG Y N, GAO L G, LIN M, et al.Recombinant expression of antimicrobial peptides in Pichia pastoris:A strategy to inhibit the Penicillium expansum in pears[J].Postharvest Biology and Technology, 2021, 171:111298.
[45] AHMAD B, HANIF Q, WEI X B, et al.In vitro impact of yeast expressed hybrid peptide CATH-2TP5 as a prophylactic measure toward sepsis and inflammation[J].Frontiers in Bioengineering and Biotechnology, 2020, 8:454.
[46] LI X H, FAN Y, LIN Q, et al.Expression of chromogranin A-derived antifungal peptide CGA-N12 in Pichia pastoris[J].Bioengineered, 2020, 11(1):318-327.
[47] NIKPOOR M, LOHRASBI-NEJAD A, ZOLALA J.Heterologous expression and functional characterization of CAP18 from Oryctolagus cuniculus[J].Reports of Biochemistry & Molecular Biology, 2022, 10(4):622-632.
[48] ZHAN N, ZHANG L C, YANG H, et al.Design and heterologous expression of a novel dimeric LL37 variant in Pichia pastoris[J].Microbial Cell Factories, 2021, 20(1):143.
[49] KJELDSEN A, KAY J E, BAXTER S, et al.The fluorescent protein iLOV as a reporter for screening of high-yield production of antimicrobial peptides in Pichia pastoris[J].Microbial Biotechnology, 2022, 15(7):2126-2139.
[50] ZHANG X Y, XI Z W, ZHAO H T, et al.Efficient heterologous expression of bovine lactoferrin in Pichia pastoris and characterization of antibacterial activity[J].Systems Microbiology and Biomanufacturing, 2025, 5(1):237-248.
[51] 王莲哲, 江宏浩, 唐宜飞, 等.新型抗菌肽Temporin-SHf在毕赤酵母中的表达及诱导条件优化[J].食品与发酵工业, 2020, 46(14):98-102.
WANG L Z, JIANG H H, TANG Y F, et al.Expression of novel antibacterial peptide Temporin-SHf in Pichia pastoris and optimization of induction conditions[J].Food and Fermentation Industries, 2020, 46(14):98-102.
[52] 李天阳, 张爱忠, 李俊, 等.含抗菌肽CC31毕赤酵母工程菌培养基及其培养条件的优化[J].中国生物制品学杂志, 2020, 33(2):197-202.
LI T Y, ZHANG A Z, LI J, et al.Optimization of culture medium and culture condition for recombinant Pichia pastoris containing CC31[J].Chinese Journal of Biologicals, 2020, 33(2):197-202.
[53] MENG D M, LI W J, SHI L Y, et al.Expression, purification and characterization of a recombinant antimicrobial peptide Hispidalin in Pichia pastoris[J].Protein Expression and Purification, 2019, 160:19-27.
[54] LI H M, ALI Z, LIU X L, et al.Expression of recombinant tachyplesin I in Pichia pastoris[J].Protein Expression and Purification, 2019, 157:50-56.
[55] POPA C, SHI X Q, RUIZ T, et al.Biotechnological production of the cell penetrating antifungal PAF102 peptide in Pichia pastoris[J].Frontiers in Microbiology, 2019, 10:1472.
[56] LAN J, MA Q Y, LI J Z, et al.Expression of T9W in Pichia pastoris and the protective roles of T9W in ICR Mice[J].Biotechnology Letters, 2020, 42(1):67-78.
[57] 张瑞. 抗菌肽Turgencin A在毕赤酵母中重组表达及其抗菌活性特性研究[D].天津:天津科技大学, 2023.
ZHANG R.Recombinant expression of antimicrobial peptide Turgencin A in saccharomyces cerevisiae and characterization of its antibacterial activity[D].Tianjin:Tianjin University of Science and Technology, 2023.
[58] 虞维红. 利用荧光蛋白做分子信标的非甲醇诱导表达PR39高表达毕赤酵母菌株的构建和筛选[D].广州:暨南大学, 2019.
YU W H.Construction and selection of non-methanol induction Pichia pastoris strain with high expression PR39 using fluorescentprotein as molecular beacon[D].Guangzhou:Jinan University, 2019.
[59] 林明, 姜路花, 余挺.Ace-AMP1过表达毕赤酵母工程菌株的构建及其对梨青霉病的抑制作用[J].浙江大学学报(农业与生命科学版), 2019, 45(1):39-46.
LIN M, JIANG L H, YU T.Overexpression of Ace-AMP1 in Pichia pastoris on enhancing the inhibition of blue mold on pears[J].Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(1):39-46.
[60] 于婷. 杂合抗菌肽Me-HNP1基因设计及其在毕赤酵母中的表达[D].长春:吉林农业大学, 2020.
Yu T.Design of the hybrid antibacterial peptide Me-HNP1 gene and its expression in Pichia pastoris[D].Changchun:Jilin Agricultural University, 2020.
[61] KRÜGER M K, SØRENSEN M A.Aminoacylation of hypomodified tRNAGlu in vivo[J].Journal of Molecular Biology, 1998, 284(3):609-620.
[62] 钊倩倩, 张秀华, 刘飞.人组织因子的密码子优化及其在毕赤酵母中的高水平发酵制备[J].药物生物技术, 2021, 28(1):1-5.
ZHAO Q Q, ZHANG X H, LIU F.Codon optimization and high level fermentation of human tissue factor in Pichia pastoris[J].Pharmaceutical Biotechnology, 2021, 28(1):1-5.
[63] 王艳慧, 陶妍.三疣梭子蟹PtCrustin2抗菌肽基因优化及其在毕赤酵母中高效表达[J].生物学杂志, 2019, 36(4):42-46.
WANG Y H, TAO Y.Gene optimization for swimming crab PtCrustin2 antimicrobial peptide and its efficient expression in Pichia pastoris[J].Journal of Biology, 2019, 36(4):42-46.
[64] WANG Y X, JIANG S, JIANG X X, et al.Cloning and codon optimization of a novel feline interferon omega gene for production by Pichia pastoris and its antiviral efficacy in polyethylene glycol-modified form[J].Virulence, 2022, 13(1):297-309.
[65] 李会宣, 董向峰, 高健.Pichia pastoris醇氧化酶基因AOX1启动子研究进展[J].生物技术, 2013, 23(4):83-87.
LI H X, DONG X F, GAO J.Progress in promoter of alcohol oxidase gene aox1 from Pichia pastoris[J].Biotechnology, 2013, 23(4):83-87.
[66] WATERHAM H R, DIGAN M E, KOUTZ P J, et al.Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter[J].Gene, 1997, 186(1):37-44.
[67] CHE Z Q, CAO X Y, CHEN G G, et al.An effective combination of Codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris)[J].BMC Biotechnology, 2020, 20(1):63.
[68] PENG Y H, WANG Y P, LIU X Y, et al.Expression and surface display of an acidic cold-active chitosanase in Pichia pastoris using multi-copy expression and high-density cultivation[J].Molecules, 2022, 27(3):800.
[69] LIU C, GONG J S, SU C, et al.Increasing gene dosage and chaperones co-expression facilitate the efficient dextranase expression in Pichia pastoris[J].LWT, 2023, 181:114753.
[70] DAI W L, DONG H F, ZHANG Z K, et al.Enhancing the heterologous expression of a thermophilic endoglucanase and its cost-effective production in Pichia pastoris using multiple strategies[J].International Journal of Molecular Sciences, 2023, 24(19):15017.
[71] DUAN G D, DING L M, WEI D S, et al.Screening endogenous signal peptides and protein folding factors to promote the secretory expression of heterologous proteins in Pichia pastoris[J].Journal of Biotechnology, 2019, 306:193-202.
[72] FULLER R S, STERNE R E, THORNER J.Enzymes required for yeast prohormone processing[J].Annual Review of Physiology, 1988, 50:345-362.
[73] 张娜, 闫亚茹, 武运, 等.信号肽优化提高葡萄糖氧化酶在毕赤酵母中的表达量[J].中国农业科技导报, 2023, 25(2):211-219.
ZHANG N, YAN Y R, WU Y, et al.Signal peptide optimization increases glucose oxidase expression in Pichia pastoris[J].Journal of Agricultural Science and Technology, 2023, 25(2):211-219.
[74] UTAMI N, NURDIANI D, HARIYATUN H, et al.Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris:A comparison[J].Journal of Genetic Engineering and Biotechnology, 2023, 21(1):67.
[75] 梁启星, 石竟成, 金学荣, 等.肠激酶在毕赤酵母中的分泌表达优化[J].生物工程学报, 2020, 36(8):1689-1698.
LIANG Q X, SHI J C, JIN X R, et al.Optimization of enterokinase secretion in Pichia pastoris[J].Chinese Journal of Biotechnology, 2020, 36(8):1689-1698.
[76] LIU C, GONG J S, SU C, et al.Pathway engineering facilitates efficient protein expression in Pichia pastoris[J].Applied Microbiology and Biotechnology, 2022, 106(18):5893-5912.
[77] VIJAYAKUMAR V E, VENKATARAMAN K.A systematic review of the potential of Pichia pastoris (Komagataella phaffii) as an alternative host for biologics production[J].Molecular Biotechnology, 2024, 66(7):1621-1639.
[78] WANG Y S, WANG B Q, GAO Y H, et al.Highly efficient expression and secretion of human lysozyme using multiple strategies in Pichia pastoris[J].Biotechnology Journal, 2023, 18(11):2300259.
[79] JIANG L X, GUAN X, LIU H J, et al.Improved production of recombinant carboxylesterase FumDM by co-expressing molecular chaperones in Pichia pastoris[J].Toxins, 2023, 15(2):156.
[80] HUANG J J, ZHAO Q Y, CHEN L X, et al.Improved production of recombinant Rhizomucor miehei lipase by coexpressing protein folding chaperones in Pichia pastoris, which triggered ER stress[J].Bioengineered, 2020, 11(1):375-385.
[81] SAMUEL P, PRASANNA VADHANA A K, KAMATCHI R, et al.Effect of molecular chaperones on the expression of Candida antarctica lipase B in Pichia pastoris[J].Microbiological Research, 2013, 168(10):615-620.
[82] ESKANDARI A, NEZHAD N G, LEOW T C, et al.Current achievements, strategies, obstacles, and overcoming the challenges of the protein engineering in Pichia pastoris expression system[J].World Journal of Microbiology & Biotechnology, 2023, 40(1):39.
[83] KHABBAZ H, KARIMI-JAFARI M H, SABOURY A A, et al.Prediction of antimicrobial peptides toxicity based on their physico-chemical properties using machine learning techniques[J].BMC Bioinformatics, 2021, 22(1):549.
[84] LOURENÇO A L P, RIOS T B, DA SILVA Á P, et al.Peptide stapling applied to antimicrobial peptides[J].Antibiotics, 2023, 12(9):1400.
[85] MIGOŃ D, NEUBAUER D, KAMYSZ W.Hydrocarbon stapled antimicrobial peptides[J].The Protein Journal, 2018, 37(1):2-12.
[86] CRESTI L, CAPPELLO G, PINI A.Antimicrobial peptides towards clinical application-a long history to be concluded[J].International Journal of Molecular Sciences, 2024, 25(9):4870.
[87] 肖浩然. 一种抗菌肽M4的设计及应用[D].成都:电子科技大学, 2024.
XIAO H R.Design and application of an antimicrobial peptide M4.Chengdu:University of Electronic Science and Technology of China, 2024.
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