|本期目录/Table of Contents|

[1]王晓飞,彭 会,陈芳奕,等.拟穴青蟹抗菌肽Crustin新变体的表达特性与抗菌功能[J].厦门大学学报(自然科学版),2019,58(03):358-365.[doi:10.6043/j.issn.0438-0479.201808007]
 WANG Xiaofei,PENG Hui,CHEN Fangyi,et al.Characterization of expression and antimicrobial activity of a novel Crustin isoform from Scylla paramamosain[J].Journal of Xiamen University(Natural Science),2019,58(03):358-365.[doi:10.6043/j.issn.0438-0479.201808007]





Characterization of expression and antimicrobial activity of a novel Crustin isoform from Scylla paramamosain
王晓飞1彭 会2陈芳奕2张财亮1黄文树13*
1.集美大学水产学院,福建 厦门 361021; 2.厦门大学海洋与地球学院,福建 厦门 361102; 3.福建省海洋生物资源开发利用协同创新中心,福建 厦门 361021
WANG Xiaofei1PENG Hui2CHEN Fangyi2ZHANG Cailiang1HUANG Wenshu13*
1.College of Fisheries,Jimei University,Xiamen 361021,China; 2.College of Ocean and Earth Sciences,Xiamen University,Xiamen 361102,China; 3.Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources,Xiamen 361021,China
拟穴青蟹 Crustin cDNA克隆 抗菌活性 扫描电镜
Scylla paramamosain Crustin cDNA cloning antimicrobial activity scanning electron microscopy
Q 789
Crustins是一类较早发现并广泛分布在甲壳动物中且富含半胱氨酸的阳离子抗菌肽,能够参与抗菌免疫应答.从拟穴青蟹(Scylla paramamosain)中克隆获得一个Crustin基因新变体,命名为SpCrus1a,其cDNA序列全长744 bp,开放阅读框编码113个氨基酸,成熟肽分子质量10.03 ku,理论等电点8.30.表达分析结果发现其转录本主要存在于鳃、卵巢、上皮组织中,经脂多糖刺激后SpCrus1a表达会上调.体外合成SpCrus1a的第29~47位氨基酸(SpCrus1a29-47)进行抗菌活性实验,发现其对革兰氏阳性菌具有较强的抗菌活性,而对被测的革兰氏阴性菌抗菌活性较弱或无抗菌活性.浓度24 μmol/L的合成肽SpCrus1a29-47能够在5 min内杀死大多数的金黄色葡萄球菌(Staphylococcus aureus),在120 min内杀死全部的金黄色葡萄球菌.扫描电镜分析发现合成肽SpCrus1a29-47可造成金黄色葡萄球菌表面结构崎岖不平,高浓度SpCrus1a29-47会引起细菌大量死亡.上述结果表明SpCrus1a是抗菌肽Crustin的新变体.
Crustins are cationic cysteine-rich antimicrobial peptides discovered earlier and widely distributed across the crustaceans,and involved in the antimicrobial immune responses.In this study,a novel Crustin isoform was cloned from Scylla paramamosain,namely SpCrus1a.The full-length cDNA of SpCrus1a was 744 bp,with an open reading frame encoding a peptide of 113 amino acids.The predicted molecular mass of the mature protein was 10.03 ku with an estimated pI of 8.30.The transcriptional transcript of SpCrus1a was highly expressed in gills,ovary and carapace by RT-PCR analysis,and was up-regulated in gills after lipopolysaccharide challenge.The antimicrobial activity experiment was performed with the synthetic peptide from 29 to 47 amino acid residues of SpCrus1a(SpCrus1a29-47).It was found that the synthetic peptide showed more strong anti-bacterial activity against Gram-positive bacteria,but had no or little antimicrobial activity against selected Gram-negative bacteria.Most of Staphylococcus aureus were killed by the synthetic peptide SpCrus1a29-47(24 μmol/L)within 5 min and all were killed within 120 min.Observation by scanning electron microscopy revealed that the surface of S. aureus cells treated with the synthetic peptide SpCrus1a29-47appeared to be rough and distorted,and higher concentration of SpCrus1a29-47 could cause cell death.The above results demonstrate that SpCrus1a is a novel Crustin isoform.


[1] WALSH C.Where will new antibiotics come from?[J].Nature Reviews Microbiology,2003,1(1):65-70.
[2] COATES A,HU Y M,BAX R,et al.The future challenges facing the development of new antimicrobial drugs[J].Nature Reviews Drug Discovery,2002,1(11):895-910.
[3] S?RENREN O E,BORREGAARD N,COLE A M.Antimicrobial peptides in innate immune responses[M]∥Contributions to microbiology.Basel:KARGER,2008:61-77.
[4] BOMAN H G,STEINER H.Humoral immunity in Cecropia pupae[J].Current Topics in Microbiology & Immunology,1981,95(4):75-91.
[5] AVITABILE C,CAPPARELLI R,RIGANO M M,et al.Antimicrobial peptides from plants:stabilization of the γ core of a tomato defensin by intramolecular disulfide bond[J].Journal of Peptide Science,2013,19(4):240-245.
[6] HASSAN M,KJOS M,NES I F,et al.Natural antimicrobial peptides from bacteria:characteristics and potential applications to fight against antibiotic resistance[J].Journal of Applied Microbiology,2012,113(4):723-736.
[7] SCHNAPP D,KEMP G D,SMITH V J.Purification and characterization of a proline-rich antibacterial peptide,with sequence similarity to Bactenecin-7,from the haemocytes of the shore crab,Carcinus maenas[J].European Journal of Biochemistry,1996,240(3):532-539.
[8] RELF J M,CHISHOLM J R S,KEMP G D,et al.Purification and characterization of a cysteine-rich 11.5-kDa antibacterial protein from the granular haemocytes of the shore crab,Carcinus maenas[J].European Journal of Biochemistry,1999,264(2):350-357.
[9] SMITH V J,FERNANDES J M O,KEMP G D,et al.Crustins:enigmatic WAP domain-containing antibacterial proteins from crustaceans[J].Developmental & Comparative Immunology,2008,32(7):758-772.
[10] CHEN J Y,CHUANG H,PAN C Y,et al.cDNA sequence encoding an antimicrobial peptide of chelonianin from the tiger shrimp Penaeus monodon[J].Fish & Shellfish Immunology,2005,18(2):179-183.
[11] CHEN D D,HE N H,XU X.Mj-DWD,a double WAP domain-containing protein with antiviral relevance in Marsupenaeus japonicus[J].Fish & Shellfish Immunology,2008,25(6):775-781.
[12] DU Z Q,REN Q,ZHAO X F,et al.A double WAP domain(DWD)-containing protein with proteinase inhibitory activity in Chinese white shrimp,Fenneropenaeus chinensis[J].Comparative Biochemistry & Physiology Part B:Biochemistry and Molecular Biology,2009,154(2):203-210.
[13] CUI Z X,SONG C W,LIU Y,et al.Crustins from eyestalk cDNA library of swimming crab Portunus trituberculatus:molecular characterization,genomic organization and expression analysis[J].Fish & Shellfish Immunology,2012,33(4):937-945.
[14] JIRAVANICHPAISAL P,LEE S Y,KIM Y A,et al.Antibacterial peptides in hemocytes and hematopoietic tissue from freshwater crayfish Pacifastacus leniusculus:characterization and expression pattern[J].Developmental & Comparative Immunology,2007,31(5):441-455.
[15] CHRISTIE A E,RUS S,GOINEY C C,et al.Identification and characterization of a cDNA encoding a crustin-like,putative antibacterial protein from the American lobster Homaru samericanus[J].Molecular Immunology,2007,44(13):3333-3337.
[16] JIA Y P,SUN Y D,WANG Z H,et al.A single whey acidic protein domain(SWD):containing peptide from fleshy prawn with antimicrobial and proteinase inhibitory activities[J].Aquaculture,2008,284(1/2/3/4):246-259.
[17] ZHANG Z T,ZHU S Y.Comparative genomics analysis of five families of antimicrobial peptide-like genes in seven ant species[J].Developmental & Comparative Immunology,2012,38(2):262-274.
[18] IMJONGJIRAK C,AMPARYUP P,TASSANAKAJON A,et al.Molecular cloning and characterization of crustin from mud crab Scylla paramamosain[J].Molecular Biology Reports,2009,36(5):841-850.
[19] WANG H,ZHANG J X,WANG Y,et al.Newly identified type Ⅱ crustin(SpCrus2)in Scylla paramamosain contains a distinct cysteine distribution pattern exhibiting broad antimicrobial activity[J].Developmental & Comparative Immunology,2018,84:1-13.
[20] WANG Y,ZHANG X W,WANG H,et al.SpCrus3 and SpCrus4 share high similarity in mud crab(Scylla paramamosain)exhibiting different antibacterial activities[J].Developmental & Comparative Immunology,2018,82:139-151.
[21] WANG Y,ZHANG C,WANG H,et al.Involvement of a newly identified atypical type Ⅱ crustin(SpCrus5)in the antibacterial immunity of mud crab Scylla paramamosain[J].Fish & Shellfish Immunology,2018,75:346-356.
[22] RATTANACHAI A,HIRONO I,OHIRA T,et al.Cloning of kuruma prawn Marsupenaeus japonicus crustin-like peptide cDNA and analysis of its expression[J].Fisheries Science,2004,70(5):765-771.
[23] SPERSTAD S V,HAUG T,PAULSEN V,et al.Characterization of crustins from the hemocytes of the spider crab,Hyas araneus,and the red king crab,Paralithodes camtschaticus[J].Developmental & Comparative Immunology,2009,33(4):583-591.
[24] YUE F,PAN L Q,MIAO J J,et al.Molecular cloning,characterization and mRNA expression of two antibacterial peptides:crustin and anti-lipopolysaccharide factor in swimming crab Portunus trituberculatus[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2010,156(2):77-85.
[25] JIANG H S,SUN C,WANG T,et al.A single whey acidic protein domain containing protein(SWD)inhibits bacteria invasion and dissemination in shrimp Marsupenaeus japonicus[J].Fish & Shellfish Immunology,2013,35(2):310-318.
[26] LATHAM P W.Therapeutic peptides revisited[J].Nature Biotechnology,1999,17(8):755-757.
[27] KRUSONG K,POOLPIPAT P,SUPUNGUL P,et al.A comparative study of antimicrobial properties of CrustinPm1 and CrustinPm7 from the black tiger shrimp Penaeus monodon[J].Developmental & Comparative Immunology,2012,36(1):208-215.


收稿日期:2018-08-12 录用日期:2018-10-24
基金项目:国家自然科学基金(U1805233,U1205123,31172438); 福建省自然科学基金(2018J01452)
更新日期/Last Update: 1900-01-01