鸭β-防御素5基因的分离、鉴定及其生物学作用

Isolation, characterization, and determination on bioactivity of duck avian beta-defensin 5

摘要:

为克隆与表达鸭β-防御素5 (AvBD5)基因及测定其生物学特性, 采用RT-PCR方法从鸭肺脏组织中扩增到鸭AvBD5, 将测得的序列与已发现的禽β-防御素和部分哺乳类动物β-防御素的氨基酸序列构建进化树进行同源性分析。结果显示鸭AvBD5 cDNA大小为201 bp, 编码66个氨基酸残基, 内含6个位置保守的半胱氨酸残基, 分别在分子内形成Cys1?Cys5、Cys2?Cys4和Cys3?Cys6共3对二硫键, 与鸡AvBD5氨基酸同源性高达97%。进一步将其亚克隆到大肠杆菌pGEX-6p-1载体中进行原核表达。重组GST融合蛋白经纯化后, 通过菌落计数法测定其体外抗菌活性, 盐离子浓度对其抗菌活性的影响, 以及该重组GST融合蛋白对溶血活性的影响。鸭AvBD5重组蛋白分子量约为32 kD, 具有良好的抗菌活性, 对11种细菌均有不同程度的抑制作用, 高盐浓度对其抗菌活性有一定影响。此外, 该重组蛋白对红细胞溶血活性极低。

Abstract:

To clone, express and characterize the antimicrobial activity of duck AvBD5, reverse transcription polymerase chain reaction (RT-PCR) was used to amplify duck AvBD5 gene from lung of duck in the present study. Furthermore, phylogenetic relationships of the duck AvBD5 with AvBDs from other avian species and some mammalian beta-defensin 5 were analyzed. Sequence analysis showed that the cDNA of duck AvBD5 consisted of 201 bp nucleotides, encoding a polypeptide of 66 amino acids. The β-defensin has six conserved cysteines that form three disulfide bonds in a C1-C5, C2-C4, and C3-C6 conformation. It was demonstrated that duck AvBD5 shared 97% amino acid homology with chicken AvBD5. The cDNA of duck AvBD5 was sub-cloned on pGEX-6p-1 vector to construct recombinant plasmid pGEX-duck AvBD5. The recombinant plasmid was transformed into E. coli BL21 and the bacteria were induced with IPTG. After purification, antibacterial activity of the purified recombinant protein was investigated. In addition, effect of ionic strength on the antibacterial activity, and hemolytic activity of the purified recombinant protein were investigated. It was demonstrated that a 32 kD protein in molecular weight was highly expressed. The purified recombinant duck AvBD5 exhibited extensive antimicrobial activity against 11 strain bacteria, including Gram-positive and Gram-negative investigated. In high salt ions conditions, antibacterial activity of recombinant duck AvBD5 protein was decreased. In addition, the recombinant protein of hemolysis activity was extremely low.