鹅β-防御素10基因的分离、鉴定与表达

Isolation,Characterization,and Expression of Goose Avian β-Defensin 10

【目的】从鹅的组织中克隆鹅β-防御素10(avianβ-defensin 10,AvBD10)基因,通过大肠杆菌进行原核表达,检测重组鹅AvBD10蛋白的生物学特性。【方法】采用RT-PCR方法,从鹅的肾脏组织中扩增鹅AvBD10基因。根据已发现的禽β-防御素和部分哺乳类动物β-防御素的氨基酸序列构建系统进化树,进行遗传进化分析;并应用荧光定量的方法对该基因的组织表达水平进行鉴定。将该基因克隆到原核表达载体pGEX-6p-1上进行原核表达,并对该重组蛋白进行纯化,测定体外生物学活性。【结果】从鹅肾脏组织中克隆出了鹅AvBD10基因,该基因的cDNA大小为207 bp,编码68个氨基酸。经遗传进化分析表明,该基因推导的氨基酸序列与鸭AvBD10的同源性最高,达92.7%。通过对重组蛋白抗菌活性的检测发现,该蛋白对12种致病性细菌均具有较强抑菌作用,但在高盐离子浓度下活性减弱。并且重组蛋白不具有溶血的特性。【结论】成功克隆并表达了鹅AvBD10基因,原核表达产物具有广谱的抗菌活性且不具有溶血特性。

【Objective】 The objective of this study was to clone avian β-defensin 10(AvBD10) gene from goose tissues and to characterize the antimicrobial activity of the recombinant GST-AvBD10 fusion protein.【Method】 The coding sequences of goose AvBD10 was obtained from kidney of goose by RT-PCR,phylogenetic relationships of the goose AvBD10 with those of other avian species and some mammalian were analyzed.In addition,tissue distribution of the gene was detected by real time PCR.AvBD gene coding sequence was inserted into the pGEX-6p-1 vector.The constructs were transformed into competent Escherichia coli BL21(DE3) cells.Expression of the fusion proteins was induced with isopropyl β-D-1-thiogalactopyranoside(IPTG) and the proteins were purified.Furthermore,bioactivities of the recombinant protein were analyzed in vitro.【Result】 The full length cDNA of goose AvBD10 consisted of 207 nucleotide acids,encoding 68 amino acid residues.Phylogenetic analysis demonstrated that the goose AvBD10 shared the highest amino acid homology with duck AvBD10(92.7%).The recombinant protein exhibited high antimicrobial activity against 12 bacterial strains investigated.The antimicrobial activity was decreased in high salt concentration and exhibited no hemolytic properties.【Conclusion】 The goose AvBD10 gene from goose was successfully cloned and expressed in E.coli.The purified recombinant protein showed antimicrobial activity and no hemolytic properties.