鸽β-防御素1基因的克隆及其生物学作用鉴定

Cloning and bioactivity determination of pigeon avian beta-defensin 1

摘要:

【目的】从鸽子组织中克隆鸽子β-防御素1 (AvBD1)基因，在大肠杆菌中表达重组鸽子AvBD1蛋白，测定其生物学特性。【方法】应用RT-PCR法从鸽子骨髓组织中扩增鸽子AvBD1基因，采用Real-time PCR法检测该基因在鸽子组织器官中的表达分布。将该基因亚克隆到大肠杆菌原核表达载体pProEX-HTa的EcoR I和Xho I双酶切位点上，构建重组表达质粒pProEX-pigeon AvBD1，将重组质粒进行诱导表达；对该重组蛋白进行纯化，通过菌落计数法测定其体外抗菌活性与理化特性。【结果】从鸽子骨髓组织中克隆到鸽子AvBD1基因，其cDNA大小为198 bp，编码65个氨基酸，经序列相似性分析，鸽子AvBD1与鸭AvBD1氨基酸序列相似性最高(81.5%)。鸽子AvBD1主要分布于免疫系统和消化系统组织中。Tricine-SDS-PAGE电泳结果表明，重组鸽子AvBD1蛋白分子量约8.8 kD，与预期大小一致。该重组蛋白具有广谱抗菌活性，高盐浓度显著降低其抗菌活性。此外，该重组蛋白的溶血活性极低。【结论】从鸽子骨髓组织中克隆到鸽子AvBD1基因，其主要分布在机体的免疫系统和消化系统中。该重组蛋白具有广谱抗菌活性，高盐浓度显著降低其抗菌活性，且该重组蛋白的溶血活性极低。

Abstract:

[Objective] The study was conducted to clone avian beta-defensin1 (AvBD1) gene from pigeon tissues, expression of recombinant AvBD1 protein in Escherichia coli and determine its biological characteristics. [Methods] The AvBD1 gene was amplified from bone marrow of pigeon by RT-PCR, differential mRNA expression of this gene was evaluated by real-time PCR. The cDNA of pigeon AvBD1 was sub-cloned into EcoR I and Xho I sites of pProEX-HTa vector to construct recombinant plasmid pProEX-pigeon AvBD1, the recombinant plasmid was expressed into E. coli, the bacteria induced with IPTG. The recombinant protein was purified, antimicrobial activity, physio-chemical characteristics of the recombinant protein were measured by colony counting assays in vitro. [Results] Sequence analysis showed that the cDNA of pigeon AvBD1 consisted of 198 bp nucleotide, encoding a polypeptide of 65 amino acids. Homology analysis showed that pigeon AvBD1 shared the highest percentage of amino acid homology (81.5%) with duck AvBD1. The mRNA was widely expressed in immune system and digestive system. It was demonstrated by Tricine-SDS-PAGE that a 8.8 kD protein which was equal to pigeon pProEX-HTa AvBD1 protein in molecular weight was expressed. The purified recombinant pigeon AvBD1 exhibit extensive antimicrobial activity. In high salt ions conditions, the antibacterial activity was significantly decreased. In addition, little hemolysis of erythrocytes was observed at any concentration of the peptide. [Conclusion] The pigeon AvBD1 gene was successfully cloned from bone marrow, the mRNA was widely expressed in immune system and digestive system. The recombinant protein exhibit extensive antimicrobial activity, has little hemolytic properties. High salt concentration significantly decreased its antibacterial activity.