本试验采用RT-PCR方法,从鹅的骨髓细胞中扩增到禽p-防御素(AvBD)基因.测序结果表明,该基因的cDNA大小为201 bp,编码66个氨基酸.经遗传进化分析发现,该基因推导的氨基酸序列与鸡AvBD5的同源性最高,达84.8％.因此,将其命名为鹅AvBD5.进一步将该基因定向插入到pGEX-6p-1的EcoR Ⅰ和SalⅠ双酶切位点上,构建pGEX-AvBD 5重组表达质粒.将重组质粒转化大肠杆菌BL 21,于37℃不同时间进行诱导表达,SDSPAGE检测外源基因的表达情况.结果表明,重组鹅AvBD 5融合蛋白的分子量约为32 ku,重组蛋白以包涵体形式存在.采用菌落计数法进行抗菌活性测定,结果表明,重组鹅AvBD 5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较强抗菌作用.此外,该蛋白对温度和pH有较高的稳定性.%In the present study, avian β-defensin (AvBD) gene was isolated from goose bone marrow tissues by RT-PCR. The complete nucleotide sequence of the gene contained a 201 bp cDNA encoding 66 amino acids. Phylogenetic relationships were analyzed. It was demonstrated that the goose AvBD shared the highest amino acid homology with chicken AvBD5 (84. 8%), and it was named as goose AvBD5. The cDNA of goose AvBD5 was sub-cloned into EcoR I and Sal I digestion sites of pGEX-6p-l vector to construct recombinant plasmid pGEX- AvBD5. The recombi-nant plasmid was transformed into E. coli BL21 and the bacterial was induced with IPTG. It was demonstrated by SDS-PAGE that a 32 ku protein was highly expressed as insoluble bodies in E. coli BL21. Colony counting assays were performed to investigate the antimicrobial activity of recombinant goose AvBD5. The result indicate that the recombinant goose AvBD5 exhibit extensive antimicrobial activity against all of bacterial strains investigated including Gram-positive and Gram-negative organisms. In addition, the results revealed that the recombinant protein retained antimicrobial activity against Pasteurella multocida under different temperatures and pH.
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