鸡β- 防御素7 的克隆及表达

    Cloning and expression of avian beta defensin 7 gene from chicken

    • 摘要: 以NCBI 已登录的鸡β- 防御素7(登录号:NM_001001194)为模板,设计特异性引物,扩增鸡β- 防御素7 基因,目的基因与pET32a 载体相连,构建表达质粒并导入大肠杆菌BL21,经IPTG 诱导表达和抗菌试验。结果表明,PCR 扩增的目的片段,经测序证明与鸡β- 防御素7 基因NM_001001194 序列同源性达99%,经IPTG 诱导表达的重组蛋白与预期大小相符。平板抑菌试验结果表明,该重组多肽对金黄色葡萄球菌有较强的抗菌活性。

       

      Abstract: A pairs of primers used in polymerase chain reaction (PCR) was designed following the NM_001001194 from GenBank to amplify the beta defensin 7 gene of chicken. The total RNA was isolated from spinal cord cells of chicken,and the first cDNA strand was obtained by approach of reverse transcription PCR. The products of PCR were linked with T plasmid vectors,and then transformed into Escherichia coli(E. Coli) DH5α strain cells subsequently. Expressional plasmid vector,pET32a-gal7,was reconstructed with restriction endonuclease Hind Ⅲ and BamH I. Recombinant plasmid vector,pET32a-gal7,was induced with Isopropyl- β-D-Thiogalacto Pyranoside (IPTG) in E. Coli DE3 strain cells to produce recombinant Gal7 protein. The results showed that the cloned cDNA sequence of beta defensin 7 gene of chicken was comprised of 132 nucleotide acid residues,and encoded the beta defensin 7 with 44 amino acids and predicted molecular weight 4923.81Da. The identity to NM_001001194 is 99%. The recombinant peptides had strong antibacterial activity against staphylococcus aureus in vitro.