鸭β-防御素4cDNA的克隆、表达及其抑菌活性的初步分析

摘要

The objective of the present study was to clone, express, characterize the antimicrobial activity of duck avian β-defensin (AvBD) 4, and determine its mRNA distribution in tissues of duck. Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify duck AvBDA gene from bursa of Fabricius of duck. Sequence analysis showed that the cDNA of duck AvBDA consisted of 171 bp nucleotide, encoding a polypeptide of 56 amino acids. Homology analysis showed that duck AvBD4 shared high percentage of amino acid homology (73. 2 %) with chicken AvBD4. The cDNA of duck AvBD4 mRNA was sub-cloned into EcoR I and Sal I sites of pGEX-6p-l vector to construct recombinant plasmid pGEX-duck AvBD4. The recombinant plasmid was transformed into E. Colt BL21 and the bacteria were induced with IPTG. It was demonstrated by SDS-PAGE that a 32 kD protein which was equal to duck AvBD4 protein in molecular weight was highly expressed.The purified recombinant duck AvBD4 exhibited extensiveantimicrobial activity against 11 strains of bacteria, including Gram-positive and Gram-negative investigated. In high salt ions conditions, recombinant duck AvBD4 protein remained antibacterial activity against Staphylococcus aureus and Pasteurella multocida. In addition, the recombinant protein of he-molytic activity was extremely low. Additionally, real time PCR was used to determine tissue distribution of the gene. It was showed that AvBD4 mRNA was expressed limitedly in tissues of ducks. It was moderately expressed in the cecal tonsil, spleen, and bursa of Fabricius of ducks.%本研究旨在克隆与表达鸭β防御素4(AvBD4)基因及测定其生物学特性,并检测其在鸭脏器组织中的分布.采用RT-PCR方法从鸭法氏囊组织中扩增到鸭AvBD4,其cDNA大小为171 bp,编码56个氨基酸.经同源性分析,鸭AvBD4与鸡AvBD4的氨基酸序列同源性最高,达73.2％.将该基因亚克隆到原核表达载体pGEX-6p-1的EcoR Ⅰ和Sal Ⅰ双酶切位点上,构建重组表达质粒pGEX-duck AvBD4.将重组质粒转化大肠杆菌BL21,于37℃进行诱导表达,SDS-PAGE电泳表明,重组鸭AvBD4蛋白的相对分子质量约为32 ku,与预期大小一致.该重组蛋白经纯化后测定其体外抗菌活性与理化特性,结果显示,重组鸭AvBD4蛋白具有广谱的抗菌活性,对11种革兰氏阳性菌和革兰氏阴性菌均具有抑菌作用.在高盐离子条件下,重组鸭AvBD4蛋白仍具有抑制金黄色葡萄球菌和多杀性巴氏杆菌的作用.此外,该重组蛋白的溶血活性极低.运用实时荧光定量PCR法检测到该基因在鸭组织器官中表达局限,仅在盲肠扁桃体、脾脏和法氏囊中表达.