鹅β-防御素3基因的分离、鉴定及其表达产物的生物学特性

Isolation,identification and bioactivity characterization of goose avian β-defensin 3

为了克隆鹅β-防御素(AvBD)3基因,并在原核表达重组鹅AvBD3蛋白,进一步研究鹅AvBD3蛋白的生物学特性,利用RT-PCR方法从鹅脾脏和法氏囊组织中扩增到鹅AvBD3基因片段,其cDNA片段大小为182 bp,编码60个氨基酸残基。经同源性分析发现鹅AvBD3氨基酸序列与鸡AvBD3氨基酸序列同源性最高,为100%。将该基因亚克隆到原核表达载体pGEX-6p-1的BamHⅠ和SalⅠ双酶切位点上,构建重组表达质粒pGEX-goose AvBD3。将重组质粒转化大肠杆菌BL21,于37℃用IPTG诱导表达,SDS-PAGE电泳表明,重组鹅AvBD3蛋白在原核高效表达(分子量约31 kDa)。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,重组鹅AvBD3蛋白具有广谱的抗菌活性,对12种细菌,包括革兰氏阳性菌和革兰氏阴性菌均具有抑菌作用。高盐离子浓度显著降低重组鹅AvBD3蛋白的抗菌活性。此外,该重组蛋白的溶血活性极低,并对酸碱度具有较高的稳定性。

The objective of the study was to clone avian 13-defensin （AvBD） 3 gene from goose tissues, express the recombinant AvBD3 protein in Escherichia coli, and determine its antimicrobial activity. The mRNA of goose AvBD3 was cloned from spleen and bursa of Fabrieius of the gooses by RT-PCR. The sequence analysis showed that the genefragment of AvBD3 contained 182 bp, and encoded 60 amino acids. Homology analysis showed that goose AvBD3 shared the highest percentage of amino acid homology （100%） with chicken AvBD3. The eDNA of goose AvBD3 was sub-cloned into BamH I and Sal I sites of pGEX-6p-1 vector toconstruct recombinant plasmid pGEX-goose AvBD3. The recombinant plasmid was transformed into E. coli BL21 and the bacteria was induced with IPTG. It was demonstrated by SDS-PAGE that a 31 kDa protein which was equal to goose AvBD3 protein in molecular weight was highly expressed. The purified recombinant goose AvBD3 exhibited extensive antimicrobial activity against twelve bacteria strains, including Gram-positive and Gram-negative investigated. At high salt ions conditions, antimicrobial activity of recombinant goose AvBD3 protein against both Staphylococcus aureus and Pastewwlla multocida decreased significantly. In addition, hemolysis activity of the recombinant protein was extremely low, and the recombinant protein remained antimicrobial activity under different pH values.