鸭β-防御素2基因的克隆、表达和表达产物的生物学特性分析

 【目的】从鸭组织中克隆鸭β-防御素（AvBD）2基因,在大肠杆菌中高效表达,检测重组鸭AvBD2蛋白的生物学特性。【方法】应用RT-PCR技术从鸭胰腺组织中扩增鸭AvBD2基因,根据已发现的禽β-防御素和部分哺乳类动物β-防御素-2的氨基酸序列构建系统进化树,将该基因克隆到大肠杆菌原核表达载体pGEX-6p-1上进行原核表达,对该重组蛋白进行纯化,测定体外抗菌活性与理化特性。【结果】鸭胰腺组织中克隆出鸭AvBD2基因的cDNA大小为195 bp,编码64个氨基酸。同源性分析表明,鸭AvBD2与其它物种AvBD2同源性较高。凝胶电泳结果显示重组鸭AvBD2蛋白在原核高效表达（分子量约为32 kD）,对多杀性巴氏杆菌、金黄色葡萄球菌、枯草芽胞杆菌有较高抗菌活性,对大肠杆菌与猪霍乱沙门氏菌的抗菌活性较弱。重组鸭AvBD2蛋白对上述细菌的最小抑菌浓度为15.25～125 μg?ml-1,对猪霍乱沙门氏菌最小抑菌浓度大于400 μg?ml-1。重组鸭AvBD2蛋白在-20～100℃或pH 3～12条件下处理30 min后仍有抗金黄色葡萄球菌作用。【结论】克隆并表达了鸭AvBD2基因,表达产物具有抗菌活性,对温度和酸碱度有较高的稳定性。

Cloning Expression and Bioactivity Characterization of Duck Avian β-Defensin 2

【Objective】 The objective of the study was to clone avian β-defensin (AvBD) 2 gene from duck tissues, expression the recombinant AvBD2 protein in E.coli, and determine its antimicrobial activity. 【Method】 The mRNA of duck AvBD2 was cloned from pancreas of duck by RT-PCR. In addition, phylogenetic relationships between amino acid sequence of the duck AvBD2, AvBDs from other avian species, and some mammalian beta-defensin-2 were analyzed. The cDNA of duck AvBD2 was sub-cloned into pGEX-6p-1 vector to construct recombinant plasmid pGEX-duck AvBD2. The recombinant protein was expressed into E. coli. and purified. Antimicrobial activity and physical-chemical stability of the recombinant fusion protein was measured in vitro. 【Result】 The sequence analysis showed that the full length cDNA of duck AvBD2 consisted of 195 bp, encoding 64 amino acid residues. Homology analysis showed that duck AvBD2 shared high amino acid homology with AvBD2 from other avian species. A high expression level of the recombinant duck AvBD2 was noted in E. coli (molecular weight, 32 kD). The recombinant protein exhibited high antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, and Pasteurella multocida, low antimicrobial activity against E. coli., and Salmonella. choleraesuis. The minimal inhibitory concentration (MIC) of the recombinant protein against the above bacteria ranged from 15.25-125 μg?ml-1. The MIC of the recombinant protein against S.choleraesuis was＞400 μg?ml-1. In addition, the results revealed that the recombinant protein retained antimicrobial activity against S. aureus under different temperatures (-20℃ to 100℃) and pH values (3 to 12). 【Conclusion】The AvBD2 gene from ducks was successfully cloned, expressed in E. coli, and purified. The recombinant protein showed antimicrobial activity and retained stability under different temperatures and pH values.