畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (6): 997-1003.doi: 10.11843/j.issn.0366-6964.2014.06.019

• 基础兽医 • 上一篇    下一篇

鸡β-防御素Gal-9 cDNA的克隆、表达及其抑菌活性的研究

冯培祥1,朱梅胜1,杨莉1,檀学进2,潘福星1,齐娟3,郭妍妍3,张毅4,尹燕博1,3*   

  1. (1.青岛农业大学 动物科技学院,青岛 266109;2.青岛市畜牧兽医研究所,青岛 266100;3.青岛澳兰百特生物工程有限公司,青岛 266101;4.中国动物卫生与流行病学中心,青岛 266032)
  • 收稿日期:2013-11-20 出版日期:2014-06-23 发布日期:2014-06-23
  • 通讯作者: 尹燕博,教授,博士,E-mail:yanboyin2011@163.com
  • 作者简介:冯培祥(1988-),男,汉族,山东胶南人,硕士生,主要从事生物制品学及动物传染病学研究,E-mail:fpx198@163.com
  • 基金资助:

    “十二五”农村领域国家高技术研究发展计划(863)项目(2012AA101303);山东省现代农业产业技术体系家禽创新团队项目(SDAIT-13-011-03)

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

FENG Pei-xiang1,ZHU Mei-sheng1,YANG Li1,TAN Xue-jin2,PAN Fu-xing1,QI Juan3,GUO Yan-yan3,ZHANG Yi4,YIN Yan-bo1,3 *   

  1. (1.College of Animal Science and Technology,Qingdao Agricultural University,Qingdao 266109, China;2.Institute of Aanimal Husbandry and Veterinary,Qingdao 266100, China;3.Qingdao Oland-Better Bioengineering Co.,Ltd,Qingdao 266101, China;4.China Animal Health and Epidemiology Center,Qingdao 266032, China)
  • Received:2013-11-20 Online:2014-06-23 Published:2014-06-23

摘要:

旨在克隆鸡 β-防御素Gal-9 基因,对其预测的成熟肽基因进行原核表达,并对产物进行抑菌活性的检测。根据 GenBank发表的鸡β-防御素基因Gal-9(NM_001001611.2)设计引物,采用RT-PCR方法从鸡食道中扩增得到鸡β-防御素Gal-9基因,将该成熟肽基因克隆到原核表达载体pET-32a(+)的EcoRⅠ/XhoⅠ双酶切位点上,构建重组原核表达质粒pET-32a-Gal-9。将重组质粒转化大肠杆菌BL21(DE3),于37 ℃进行诱导表达。结果扩增出Gal-9,其cDNA为204 bp,成熟肽编码42个氨基酸。SDS-PAGE电泳表明,重组鸡Gal-9蛋白大小约为25 ku,与预期大小一致,重组蛋白主要以包涵体形式存在。重组鸡 Gal-9 蛋白对金黄色葡萄球菌(ATCC 25923)、粪肠球菌(ATCC 29212)、大肠杆菌(ATCC 25922)、酵母菌 (GS115) 都能产生抑菌作用,最小抑菌浓度分别为62.50、31.75、125.00、31.75 mg•L-1。成功获得了鸡 β-防御素Gal-9成熟肽基因的表达产物,证实了重组鸡Gal-9蛋白具有广谱的抗菌活性,体外抑菌试验为其在家禽生产中应用提供了理论基础。

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.

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