鸭β-防御素9基因的克隆、组织分布及其原核表达

doi:10.3864/j.issn.0578-1752.2009.04.034

中国农业科学 ›› 2009, Vol. 42 ›› Issue (4): 1406-1412 .doi: 10.3864/j.issn.0578-1752.2009.04.034

鸭β-防御素9基因的克隆、组织分布及其原核表达

东北农业大学动物营养研究所

收稿日期:2008-07-26 修回日期:2008-10-29 出版日期:2009-04-10 发布日期:2009-04-10
通讯作者: 马得莹

mRNA Cloning, Tissue Distribution and Expression of Duck Avian Beta-Defensin 9 in E. coli

东北农业大学动物营养研究所

Received:2008-07-26 Revised:2008-10-29 Online:2009-04-10 Published:2009-04-10
Contact: MA De-ying

摘要/Abstract

摘要：

【目的】从鸭组织中克隆鸭β-防御素9（AvBD9）基因，在原核表达重组鸭AvBD9蛋白;研究重组鸭AvBD9蛋白的体外抗菌活性与理化特性。【方法】采用RT-PCR法，从鸭肝脏组织中扩增鸭AvBD9基因，测定该基因在鸭各组织器官中的分布;将鸭AvBD9基因克隆到大肠杆菌原核表达载体pGEX-6p-1上，构建重组表达质粒pGEX-duck AvBD9，将重组质粒转化大肠杆菌BL21，用IPTG诱导表达;亲和层析纯化蛋白，利用薄层平皿琼脂糖孔穴扩散法测定重组鸭AvBD9蛋白的体外抗菌活性与理化特性。【结果】从鸭肝脏组织中克隆得到鸭AvBD9基因，序列分析表明该基因大小为204 bp，前体肽包括67个氨基酸残基。AvBD9基因在鸭体内广泛分布。SDS-PAGE电泳表明鸭AvBD9基因在大肠杆菌中高效表达，表达的重组鸭AvBD9融合蛋白以包涵体的形式存在，分子量约31 kD。重组鸭AvBD9蛋白对大肠杆菌、多杀性巴氏杆菌、金黄色葡萄球菌、嗜酸乳杆菌、枯草芽胞杆菌有抗菌活性，在-70～100℃或pH 3～10处理30 min后仍有抗多杀性巴氏杆菌作用。【结论】鸭AvBD9基因为204 bp，编码67个氨基酸残基，在鸭组织中广泛分布。重组鸭AvBD9蛋白基因在大肠杆菌中高效表达，该重组蛋白具有广谱的抗菌活性，对温度和酸碱性有较高的稳定性。

关键词: 鸭, AvBD9, 组织分布, 重组蛋白, 抗菌活性

Abstract:

【Objective】 The study was conducted to clone avian beta-defensin 9 (AvBD9) gene from duck tissues and determine tissue distribution of the gene in ducks. In addition, expression of recombinant AvBD9 protein in E. coli and determination of its antimicrobial activity were performed. 【Method】 The mRNA of duck AvBD9 was cloned from liver of duck by RT-PCR. In addition, differential mRNA expression of the gene has been demonstrated across a panel of tissues in the duck. The cDNA of duck AvBD9 mRNA was sub-cloned into EcoR I and Sal I sites of pGEX-6p-1 vector to construct recombinant plasmid pGEX-duck AvBD9. The recombinant plasmid was transformed into E. coli BL21 and the bacteria were induced with IPTG. The recombinant fusion protein was purified by affinity chromatograph. Antimicrobial activity, physio-chemical characteristics of the recombinant fusion protein were measured by inhibition zone assay. 【Result】 The sequence analysis showed that the full length cDNA of duck AvBD9 consisted of 204 bp encoding 67 amino acid residues. Duck AvBD9 mRNA was widely expressed in the tissues of duck. It was demonstrated by SDS-PAGE that a 31 kD protein which was equal to duck AvBD9 protein in molecular weight was highly expressed as insoluble bodies in E. coli BL21. The recombinant duck AvBD9 exhibited antimicrobial activity against Escherichia coli, Lactobacilli acidophilus, Bacillus subtilis, Staphylococcus aureus, and Pasteurella multocida. In addition, the results revealed that the recombinant protein retained its antimicrobial activity under different temperatures (from –70℃ to 100℃) and pH values (from 3 to 10). 【Conclusion】 The full length cDNA of duck AvBD9 consistes of 204 bp encoding 67 amino acid residues. Duck AvBD9 mRNA was widely expressed in the tissues of duck. Duck AvBD9 protein is highly expressed as insoluble bodies in E. coli BL21. The recombinant protein exhibited broad-spectrum antimicrobial activity and retained antimicrobial activity under different temperatures and pH values.

Key words: duck, AvBD9, tissues distribution, recombinant protein, antimicrobial activity

. 鸭β-防御素9基因的克隆、组织分布及其原核表达[J]. 中国农业科学, 2009, 42(4): 1406-1412 .

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鸭β-防御素9基因的克隆、组织分布及其原核表达

mRNA Cloning, Tissue Distribution and Expression of Duck Avian Beta-Defensin 9 in E. coli

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