牦牛β-防御素5基因的原核表达及表达产物的抑菌活性

Prokaryotic expression and antibacterial activity of β-defensin 5 in yak

【目的】克隆与表达牦牛β-防御素5(BNBD5)基因,检测其重组蛋白的体外抗菌活性。【方法】采用RT-PCR方法从牦牛肺组织中扩增BNBD5基因成熟肽编码区,根据已发现的哺乳动物β-防御素5和部分禽β-防御素5的序列构建遗传进化树。将BNBD5基因亚克隆到原核表达载体pET-32a(+)的BamHⅠ和XhoⅠ双酶切位点上,构建重组表达质粒pET32a-BNBD5。将重组表达质粒转化大肠杆菌BL21,用IPTG于37℃进行诱导表达,SDSPAGE检测融合蛋白的表达情况,并对该重组蛋白进行纯化,测定其体外抑菌活性。【结果】克隆得到了BNBD5基因成熟肽编码片段,其长度为138bp,编码45个氨基酸残基,内含6个位置保守的半胱氨酸残基。经遗传进化分析发现,该基因序列与黄牛的mRNA同源性最高,可达到86.2%。经IPTG诱导,获得了分子质量为25ku的牦牛β防御素-5成熟肽融合蛋白。琼脂糖扩散结果表明,0.08mg/mL的纯化蛋白对大肠杆菌及金黄色葡萄球菌均有抗菌作用。【结论】牦牛BNBD5成熟肽在大肠杆菌中得到了成功表达,其产物对革兰氏阴性菌(大肠杆菌)和革兰氏阳性菌(金黄色葡萄球菌)均有抗性。

【Objective】The purpose of this study was to clone and express yakβ-defensin 5gene(BNBD5)and to determine its antibacterial activity.【Method】The mature peptide of encoding yak BNBD5 was cloned from lung tissues by RT-PCR.In addition,phylogenetic relationships between nucleotide sequence of yak BNBD5 and BNBD5of other species were analyzed by DNAStar.The mature peptide of yak BNBD5 was sub-cloned into pET-32a(+)vector between BamHⅠ and XhoⅠsite to construct recombinant plasmid pET32-BNBD5.Then,the BNBD5 fusion protein was induced to express in BL21 by IPTG at37℃and SDS-PAGE was used to detect the expression.The expression product was purified for detection of antibacterial activity in vitro.【Result】Mature peptide of encoding yak BNBD5 was obtained with 138 bp nucleotides and 45 amino acids,including 6conserved cysteines.Phylogenetic analysis showed that yak BNBD5 shared the highest nucleotide homology(86.2%)with Bos taurus.The molecular weight of expression product was approximately 25 ku after IPTG induction.The agar diffusion method demonstrated that purified mature peptide protein with concentration of 0.08mg/mL had obvious antimicrobial activityagainst S.aureus and E.coil.【Conclusion】The mature peptide successfully expressed in E.coil and the product had resistance to both Gram negative(E.coil)and Gram positive(S.aureus)bacteria.