鸡β-防御素Gal-9 cDNA的克隆、表达及其抑菌活性的研究

doi:10.11843/j.issn.0366-6964.2014.06.019

Abstract

Abstract:

The aim of this study was to clone and express the chicken β-defensin Gal-9 gene，and detect the antibacterial activity of the expressed product.The Gal-9 gene was amplified from chicken’s esophagus by RT-PCR，with primers based on the chicken Gal-9 (NM_001001611.2) sequence in GeneBank，then it was cloned into pET-32a(+) vector using the restriction sites of EcoRⅠ/XhoⅠ.The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced at 37 ℃.The length of Gal-9 cDNA was 204 bp，and encoding 42 amino acid mature peptide.The product was confirmed to be about 25 kD in size by SDS-PAGE.It was mainly expressed in the form of inclusion body.The recombinant chicken Gal-9 protein showed bacteriostatic action on Staphylococcus aureus (ATCC 25923)，Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922)，and yeast (GS115).The minimum inhibitory concentration was 62.50，31.75，125.00，31.75 mg•L^－1.The results presented here demonstrate that the Gal-9 mature peptide gene，chicken β-defensin，was successfully expressed.The expressed protein showed broad-spectrum antimicrobial activity，which indicated the recombinant chicken Gal-9 protein could be potentially used for prevention and treatment.

CLC Number:

S852.42

FENG Pei-xiang，ZHU Mei-sheng，YANG Li，TAN Xue-jin，PAN Fu-xing，QI Juan，GUO Yan-yan，ZHANG Yi，YIN Yan-bo. cDNA Cloning，Expression and Antibacterial Activity Analysis of Chicken β-defensin Gal-9[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(6): 997-1003.

0
/ / Recommend

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

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2014.06.019

https://www.xmsyxb.com/EN/Y2014/V45/I6/997

References

［1］BRUNE K，LEFFELL M S，SPITZNAGEL J K.Microbicidal activity of peroxidaseless chicken heterophile leukocytes［J］.Infect Immun，1972，5(3)：283-287.
［2］SUGIARTO H，YU P L.Avian antimicrobial peptides：the defense role of β-defensins［J］.Biochem Biophys Res Commun，2004，323(3)：721-727.
［3］SCHUTTE B C，MCCRAY P J.β-defensins in lung host defense［J］.Annu Rev Physiol, 2002，64：709-748.http://www.annualreviews.org/doi/pdf/10.1146/annurev.physiol.64.081501.134340.
［4］WANG W，COLE A M，HONG T，et al.Retrocyclin，an antiretroviral theta-defensin，is a lectin［J］.J Immunol, 2003，170(9)：4708-4716.
［5］MA D，WANG R，LIAO W，et al.Identification and characterization of a novel antibacterial peptide，avian β-defensin 2 from ducks［J］.J Microbiol，2009，47（5）：610-618.
［6］SUGIARTO H，YU P L.Identification of three novel ostricacins：an update on the phylogenetic perspective of beta-defensins［J］.Int J Antimicrob Agents，2006，27(3)：229-235.
［7］MA D Y，LIU S W，HAN Z X，et al.Expression and characterization of recombinant gallinacin-9 and gallinacin-8 in Escherichia coil［J］.Protein Expr Purif，2008，58(2)： 284-291.
［8］MILONA P，TOWNES C L，BEVAN R M，et al.The chicken host peptides，gallinacins 4，7,and 9，have antimicrobial activity against Salmonella serovars［J］.Biochem Biophys Res Commun，2007，356(1)：169-174.
［9］张祥斌，毕英佐，曹永长，等．胡须鸡 β-防御素Gal-1~Gal-13基因克隆、序列分析及其在组织中分布［J］.农业生物技术学报，2008，16(4)：597-603．
［10］VAN D A，VELDHUIZEN E J，KALKHOVE S I，et al.The beta-defensin gallinacin-6 is expressed in the chicken digestive tract and has antimicrobial activity against food-borne pathogens ［J］.Antimicrob Agents Chemother，2007，51(3)：912-922.
［11］HUANG G T，ZHANG H B，KIM D，et al.A model for antimicrobial gene therapy：demonstration of human β-defensin 2 antimicrobial activities in vivo［J］.Hum Gene Ther，2002，13(17)：2017-2025.
［12］王璟，杨玉荣，梁宏德，等.禽类防御素作用机理及基因工程研究进展［J］.动物医学进展，2007，28(10)：81-84.
［13］LEVENGOOD M R，KNERR P J，OMAN T J，et al.In vitro mutasynthesis of lantibiotic analogues containing nonproteinogenic amino acids［J］.J Am Chem Soc，2009，131(34):12024-12025.
［14］XIAO Y，HUGHES A L，ANDO J，et al.A genome-wide screen identifies a single beta-defensin gene cluster in the chicken：implications for the origin and evolution of mammalian defensins［J］.BMC Genomics，2004，5(1)：56.
［15］周财源，蔺利娟，韩宗玺，等.鹅β-防御素基因克隆与生物学特性的初步分析［J］.畜牧兽医学报，2011，42（8）：1193-1200.
［16］HIGGS R，LYNN D J，GAINES S，et al.The synthetic form of a novel chicken beta-defensin identified in silico is predominantly active against intestinal pathogens［J］.Immunogenetics，2005，57(1-2)：90-98.
［17］GANZ T.Defensins and other antimicrobial peptides：a historical perspective and an update［J］.Comb Chem High Throughput Screen，2005，8(3)：209-217.
［18］韩宗玺，廖文艳，王瑞琴，等．重组鸡β-防御素6基因的表达和生物学特性的研究［J］.中国预防兽医学报，2009，31(6)：476-480.
［19］MA D，LIAO W，WANG R，et al.Two novel duck antibacterial peptides，avian beta-defensins 9 and 10，with antimicrobial activity ［J］.J Microbiol Biotechnol，2009，19 (11)：1447-1455.
［20］李民欢，祁克宗，涂健，等.鸡β-防御素 7 在大肠杆菌中的表达及活性分析［J］.微生物学通报，2012，39（12）：1778-1786.
［21］廖文艳，马得莹，韩宗玺，等.重组鸡β-防御素 10 蛋白的原核表达及其抗菌活性的测定［J］.中国预防兽医学报，2008，30（10）：765-769.
［22］廖文艳，马得莹，王瑞琴，等．鸭β-防御素10基因的克隆、遗传进化分析及其生物学特性的初步研究［J］.畜牧兽医学报，2009，40(9)：1320-1326.
［23］SANG Y，ORTEGA M T，BLECHA F，et al.Molecular cloning and characterization of three beta-defensins from canine testes［J］.Infect Immun，2005，73(5)：2611-2620．
［24］王威，王联结．阳离子抗菌肽分子设计的研究现状［J］.食品工业科技，2010，31(1)：442-445，448.

[1]	WENG Xiang, ZHANG Ailian, LI Quanxiao, WU Daocheng, CAO Hui. Immuno-enhancement Effects of Cultivated Artemisia rupestris L. Crude Polysaccharide on Foot-and-mouth Disease Vaccine via Subcutaneous Route [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 315-323.
[2]	QIAN Man, LIAO Chengshui, ZHANG Chunjie. Research Progress on Extracellular Traps of Innate Immune Cells Stimulated by Foodborne Pathogens [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(7): 1800-1808.
[3]	SONG Yinjuan, LIAO Yi, ZHOU Xiangmei. The Role of Mitochondrial DNA in Innate Immunity [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(2): 286-299.
[4]	LI Nan, ZHANG Jianlou, ZHANG Yonghong, ZHENG Zhiqiang, HUO Shanshan, ZHONG Fei, ZHAI Xianghe. Preparation of Bovine Surfactant Protein A and Its Antibacterial Activities [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(2): 498-505.
[5]	WANG Jingyu, OUYANG Wei, QIAN Jing, WANG Xiaoli, XIA Xingxia, ZHU Yumei, BI Zhenwei, WANG Yongshan. Expression and Antiviral Activity Analysis of Canine Interferon Alpha 2 and Canine Albumin Fusion Gene in Insect Cells Expression System [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(10): 2113-2118.
[6]	LI Lan, QIAO Xu-wen, ZHANG Yuan-peng, CHEN Jin, ZHENG Qi-sheng, HOU Ji-bo. Expression of Codon-optimized Porcine Ficolin α in Insect Cell and Its Antiviral Activity in vitro [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(12): 2358-2364.
[7]	LIAO Cheng-shui，BOIREAU Pascal，LIU Ming-yuan，CHENG Xiang-chao. Research Progress on Extracellular Traps：the Third kinds of Defence Mechanisms of Innate Immune Cells [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(9): 1768-1774.
[8]	HAO Gang，LE Guo-wei，SHI Yong-hui. The Bactericidal Mechanism of Two Analogues of the Antimicrobial Peptide BuforinⅡSpecific Binding to Staphylococcus aureus Genomic DNA [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(7): 1154-1161.

cDNA Cloning，Expression and Antibacterial Activity Analysis of Chicken β-defensin Gal-9

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 8

Recommended Articles

Metrics

Comments