鹅β-防御素10基因的分离、鉴定与表达

doi:10.3864/j.issn.0578-1752.2012.05.022

Abstract

Abstract: 【Objective】 The objective of this study was to clone avian β-defensin 10 (AvBD10) gene from goose tissues and to characterize the antimicrobial activity of the recombinant GST-AvBD10 fusion protein. 【Method】 The coding sequences of goose AvBD10 was obtained from kidney of goose by RT-PCR, phylogenetic relationships of the goose AvBD10 with those of other avian species and some mammalian were analyzed. In addition, tissue distribution of the gene was detected by real time PCR. AvBD gene coding sequence was inserted into the pGEX-6p-1 vector. The constructs were transformed into competent Escherichia coli BL21 (DE3) cells. Expression of the fusion proteins was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) and the proteins were purified. Furthermore, bioactivities of the recombinant protein were analyzed in vitro. 【Result】 The full length cDNA of goose AvBD10 consisted of 207 nucleotide acids, encoding 68 amino acid residues. Phylogenetic analysis demonstrated that the goose AvBD10 shared the highest amino acid homology with duck AvBD10 (92.7%). The recombinant protein exhibited high antimicrobial activity against 12 bacterial strains investigated. The antimicrobial activity was decreased in high salt concentration and exhibited no hemolytic properties. 【Conclusion】 The goose AvBD10 gene from goose was successfully cloned and expressed in E. coli. The purified recombinant protein showed antimicrobial activity and no hemolytic properties.

Key words: goose AvBD10, clone, phylogenetic analysis, tissue distribution, antimicrobial activity

ZHOU Cai-Yuan, ZHANG Ming-Yue, HAN Zong-Xi, SHAO Yu-Hao, LIU Sheng-Wang, MA De-Ying. Isolation, Characterization, and Expression of Goose Avian β-Defensin 10[J].Scientia Agricultura Sinica, 2012, 45(5): 999-1009.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2012.05.022

https://www.chinaagrisci.com/EN/Y2012/V45/I5/999

References

[1]van Dijk A, Veldhuizen E J A, Haagsman H P. Avian defensins. Veterianary Immunology and Immunopathology, 2008, 124(1/2): 1-8.

[2]Evans E W, Beach G G, Wunderlich J, Harmon B G. Isolation of antimicrobial peptides from avian heterophils. Journal of Leukocyte Biology, 1994, 56(5): 661-665.

[3]Higgs R, Lynn D J, Gaines S, McMahon J, Tiemey J, James T, LIoyd A T, Mulcahy G, O’Farrelly C. The synthetic form of a novel chicken beta-defensin identified in silico is predominantly active against intestinal pathogens. Immunogenetics, 2005, 57(1/2): 90-98.

[4]Lynn D J, Higgs R, Gaines S, Tiemey J, James T, LIoyd A T, Fares M A, Mulcahy G, O’Farrelly C. Bioinformatic discovery and initial characterisation of nine novel antimicrobial peptide genes in the chicken. Immunogenetics, 2004, 56(3): 170-177.

[5]Ma D Y, Liao W Y, Wang R Q, Han Z X, Liu S W. Two novel duck antibacterial peptides, avian β-defensins 9 and 10, with antimicrobial activity. Journal of Microbiology and Biotechnology, 2009, 19(11): 1447-1455.

[6]Xiao Y J, Hughes A L, Ando J, Matsuda Y, Cheng J F, Skinner-Noble D, Zhang G L. A genome-wide screen identifies a single beta-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins. BMC Genomics, 2004, 5(1): 56-67.

[7]Brune K, Leffell M S, Spitznagel J K. Microbicidal activity of peroxidaseless chicken heterophile leukocyte. Infection and Immunity, 1972, 5(3): 283-287.

[8]Donovan K L, Topley N. What are renal defesins defending? Nephron Experimental Nephrology, 2003, 93(4): 125-128.

[9]Sugiarto H, Yu P L. Avian antimicrobial peptides: the defense role of beta-defensins. Biochemical and Biophysical Research Communications, 2004, 323(3): 721-727.

[10]Higgs R, Lynn D J, Gaines S, McMahon J, Tierney J, James T, LIoyd A T, Mulcahy G, O’Farrelly C. The synthetic form of a novel chicken beta-defensin identified in silico is predominantly active against intestinal pathogens. Immunogenetics, 2005, 57(1/2): 90-98.

[11]Lynn D J, Higgs R, Lloyd A T, O’Farrelly C, Herve-Grepinet V, Nys Y, Brinkman F S , Yu P L, Soulier A, Kaiser P, Zhang G, Lehrer R I. Avian beta-defensin nomenclature: a community proposed update. Immunology Letters, 2007, 110(1): 86-89.

[12]廖文艳, 马得莹, 刘胜旺, 韩宗玺. 重组鸭β-防御素9基因的克隆、组织分布及其原核表达. 中国农业科学, 2009, 42(4): 1406-1412.

Liao W Y, Ma D Y, Liu S W, Han Z X. mRNA cloning, tissues distribution and expression in E.coli of duck avian beta-defensin 9. Scientia Agriculture Sinica, 2009, 42(4): 1406-1412. (in Chinese)

[13]韩宗玺, 廖文艳, 王瑞琴, 邵昱昊, 马得莹. 重组鸭β-防御素6 基因的表达和生物学特性的研究. 中国预防兽医学报, 2009, (6): 476-480.

Han Z X, Liao W Y, Wang R Q, Shao Y H, Ma D Y. Expression and characterization of chicken avian beta-defensin 6. Chinese Journal of Preventive Veterinary Medicine, 2009, (6): 476-480. (in Chinese)

[14]王瑞琴, 廖文艳, 马得莹, 韩宗玺, 刘胜旺. 鸭β-防御素2 基因克隆、表达和表达产物的生物学特性分析. 中国农业科学, 2009, 42(10): 3685-3692.

Wang R Q, Liao W Y, Ma D Y, Han Z X, Liu S W. Cloning, expression and bioactivity characterization of duck avian beta-defensin 2. Scientia Agriculture Sinica, 2009, 42(10): 3685-3692. (in Chinese)

[15]Wang R Q, Ma D Y, Lin L J, Zhou C Y, Han Z X, Shao Y H, Liao W Y, Liu S W. Identification and characterization of an avian β-defensin orthologue, avian β-defensin 9, from quails. Applied Microbiology and Biotechnology, 2010, 87(4): 1395-1405.

[16]周财源, 蔺利娟, 韩宗玺, 邵昱昊, 刘胜旺, 马得莹. 鹅β-防御素基因克隆与生物学特性的初步分析. 畜牧兽医学报, 2011, 42(8): 1193-1200.

Zhou C Y, Lin L J, Han Z X, Shao Y H, Liu S W, Ma D Y. Cloning and initial characterization of goose avian β-defensin. Acta Veterinaria et Zootechnica Sinica, 2011, 42(8): 1193-1200. (in Chinese)

[17]Kim A. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Reviews Microbiology, 2005, (3): 238-250.

[18]Milona P, Townes C L, Bevan R M, Hall J. The chicken host peptides gallinacins 4, 7 and 9 have antimicrobial activity against salmonella serovars. Biochemical and Biophysical Research Communications, 2007, 356(1): 169-174.

[19]Sugiarto H, Yu P L. Mechanisms of action of ostrich beta-defensins against Escherichia coli. FEMS Microbiology Letters, 2007, 270: 195-200.

[20]Hughes A L. Evolutionary diversification of the mammalian defensins. Cellular and Molecular Life Sciences, 1999, 56(1/2): 94-103.

[21]Ganz T. Defensins and other antimicrobial peptides: a historical perspective and an update. Combinatorial Chemistry and High Throughput Screen, 2005, 8(3): 209-217.

[22]Ma D Y, Liu S W, Han Z X, Li Y J, Shan A S. Expression and characterization of recombinant gallinacin-9 and gallinacin-8 in Escherichia coli. Protein Expression Purification, 2008, 58(2): 284-291.

[23]Zhao C, Nguyen T, Liu L, Sacco R E, Brogden K A, Lehrer R I. Gallinacin-3, an inducible epithelial beta-defensin in the chicken. Infection and Immunity, 2001, 69(4): 2684-2691.

[24]Thouzeau C, Le Maho Y, Froget G, Sabatier L, Le Bohec C, Hoffmann J A, Bulet P. Spheniscins, avian beta-defensins in preserved stomach contents of the king penguin, Aptenodytes patagonicus. The Journal of Biological Chemistry, 2003, 278(51): 51053-51058.

[25]Harmon B G. Avian heterophils in Inflammation and disease resistance. Poultry Science, 1998, 77(7): 972-977.

[26]Ross D J, Cole A M, Yoshioka D, Park A K, Belperio J A, Laks H, Strieter R M, Lynch J P 3rd, Kubak B, Ardehali A, Ganz T. Increased bronchoalveolar lavage human beta-defensin type 2 inbronchiolitis obliterans syndrome after lung transplantation. Transplantation, 2004, 78(8): 1222-1224.

Related Articles 15

[1]	JIANG QiQi,XU JianJian,SU Yue,ZHANG Qi,CAO Peng,SONG ChenHu,LI ZhongAn,SONG Zhen. Construction and Application of Infectious Clone of Citrus Yellow Mosaic Virus [J]. Scientia Agricultura Sinica, 2022, 55(24): 4840-4850.
[2]	LI ZhengGang,TANG YaFei,SHE XiaoMan,YU Lin,LAN GuoBing,HE ZiFu. Molecular Characteristics and Pathogenicity Analysis of Youcai Mosaic Virus Guangdong Isolate Infecting Radish [J]. Scientia Agricultura Sinica, 2022, 55(14): 2752-2761.
[3]	GUO FengHui,DING Yong,JI Lei,LI XianSong,LI XiLiang,HOU XiangYang. The Response of Leymus chinensis Cloned Offspring to Mowing [J]. Scientia Agricultura Sinica, 2022, 55(11): 2257-2264.
[4]	JiaQi WANG,YuHong DONG,JuLing JIANG,JianNing QIAN,WenTao WEI,GuoLiang SONG,JinBo JIAO,XinXin GUAN,GuoBiao JI,YeXin ZHANG. Based on PK15 Cell Line for PCV2 Fully Suspension Culture Process [J]. Scientia Agricultura Sinica, 2021, 54(6): 1280-1287.
[5]	ZHANG Li,TANG YaFei,LI ZhengGang,YU Lin,LAN GuoBing,SHE XiaoMan,HE ZiFu. Molecular Characteristic of Squash Leaf Curl China Virus (SLCCNV) Infecting Cucurbitaceae Crops in Guangdong Province [J]. Scientia Agricultura Sinica, 2021, 54(19): 4097-4109.
[6]	LI ZuRen,LUO DingFeng,BAI HaoDong,XU JingJing,HAN JinCai,XU Qiang,WANG RuoZhong,BAI LianYang. Cloning and Expression Analysis of Light Harvesting Chlorophyll a/b Protein Gene CcLhca-J9 in Conyza canadensis [J]. Scientia Agricultura Sinica, 2021, 54(1): 86-94.
[7]	KunNeng ZHOU,JiaFa XIA,Peng YUN,YuanLei WANG,TingChen MA,CaiJuan ZHANG,ZeFu LI. Transcriptome Research of Erect and Short Panicle Mutant esp in Rice [J]. Scientia Agricultura Sinica, 2020, 53(6): 1081-1094.
[8]	LI ZhengGang,NONG Yuan,TANG YaFei,SHE XiaoMan,YU Lin,LAN GuoBing,DENG MingGuang,HE ZiFu. Molecular Characteristic and Pathogenicity Analyses of Cucumber green mottle mosaic virus (CGMMV) Infecting Bottle Gourd in Lianzhou, Guangdong [J]. Scientia Agricultura Sinica, 2020, 53(5): 955-964.
[9]	XU JianJian,WANG YanJiao,DUAN Yu,MA ZhiMin,BIN Yu,ZHOU ChangYong,SONG Zhen. Construction of Genome-Length cDNA of Citrus Vein Enation Virus and Identification of Its Infectivity [J]. Scientia Agricultura Sinica, 2020, 53(18): 3707-3715.
[10]	CHEN WenFeng,WANG HongFang,LIU ZhenGuo,ZHANG WeiXing,CHI XuePeng,XU BaoHua. Recombinant Expression and Antimicrobial Activity of Apidaecin in Apis cerana cerana [J]. Scientia Agricultura Sinica, 2019, 52(4): 767-776.
[11]	WANG QiuYing,WANG WeiQiao,ZHANG Yan,WANG GuoNing,WU LiQiang,ZHANG GuiYin,MA ZhiYing,YANG Jun,WANG XingFen. Cloning and Functional Characterization of Gene CRVW Involved in Cotton Resistance to Verticillium Wilt [J]. Scientia Agricultura Sinica, 2019, 52(11): 1858-1869.
[12]	CUI TianTian, YAN JianHong, BIN Yu, LI ZhongAn, ZHOU ChangYong, SONG Zhen. Construction of Citrus leaf blotch virus Infectious cDNA Clone by Yeast Homologous Recombination System [J]. Scientia Agricultura Sinica, 2018, 51(9): 1695-1705.
[13]	PU Yan, LIU Chao, LI Ji-Yang, AERZU GULI·TaShi, HU Yan, LIU XiaoDong. Different SlU6 Promoters Cloning and Establishment of CRISPR/Cas9 Mediated Gene Editing System in Tomato [J]. Scientia Agricultura Sinica, 2018, 51(2): 315-326.
[14]	XIE HaiKun, JIAO Jian, FAN XiuCai, ZHANG Ying, JIANG JianFu, SUN HaiSheng, LIU ChongHuai. Assembling and Characteristic Analysis of the Complete Chloroplast Genome of Vitis vinifera cv. Cabernet Sauvignon from High-Throughput Sequencing Data [J]. Scientia Agricultura Sinica, 2017, 50(9): 1655-1665.
[15]	LI YaBo, LI Ting, HAN YingYan, FAN ShuangXi. Cloning and Function Analysis of Heat-Shock-Protein LsHsp70-2711 Gene Under High Temperature Stress in Leaf Lettuce (Lactuca sativa L.) [J]. Scientia Agricultura Sinica, 2017, 50(8): 1486-1494.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

Isolation, Characterization, and Expression of Goose Avian β-Defensin 10

PDF