鹌鹑β-防御素10基因的克隆与表达及其体内分布的检测

摘要

为测定鹌鹑β-防御素10 (AvBD10)体外抗菌活性及检测其在鹌鹑脏器组织的分布,本研究应用RT-PCR方法从鹌鹑肺脏组织中扩增到鹌鹑AvBD10,经序列同源性分析,鹌鹑AvBD10与鸡AvBD10的氨基酸序列同源性最高(83.8％).将该基因亚克隆到表达载体pGEX-6p-1中进行原核表达,SDS-PAGE电泳表明,鹌鹑AvBD10重组蛋白分子量约为31ku.该重组蛋白经纯化后测定其体外抗菌活性,结果显示,鹌鹑AvBD10重组蛋白具有广谱的抗菌活性.采用定量RT-PCR法检测到AvBD10在鹌鹑脏器组织中广泛表达.%To clone and express avian beta-defensin (AvBDIO) gene in quail, the quail AvBDIO gene was amplified from lung of Chinese painted quail (Coturnix chinensis) by RT-PCR. Sequence analysis showed that the cDNA of quail AvBDIO consisted of 207 bp nucleotide, encoding a polypeptide of 68 amino acids. Homology analysis showed that quail AvBDIO shared 83.8% with chicken AvBDIO. The cDNA of quail AvBDIO was sub-cloned into pGEX-6p-l vector and expressed in E. Coii BL21. SDS-PAGE analysis indicated that 31 ku of quail AvBDIO recombinant protein was highly expressed. The purified recombinant quail AvBDIO exhibit extensive antimicrobial activity against 10 different bacteria. Additionally, results of real-time RT-PCR detections showed that AvBDIO was widely expressed in various quail tissues.