驯鹿β-防御素-1(reBD-1)基因的原核表达及蛋白活性的鉴定

Prokaryotic Expression of Reindeer β-defensin-1(reBD-1) Gene and Bioactivity Identification of the Recombinant Protein

旨在构建驯鹿β防御素-1(Reindeerβ-defensin-1,reBD-1)基因的原核表达载体pET-32a(+)/reBD-1,诱导reBD-1融合蛋白在大肠杆菌中表达,并对其表达产物的生物学活性进行评价。利用RT-PCR技术扩增reBD-1前原肽。从重组克隆载体PMD19T/reBD-1中扩增reBD-1成熟肽编码基因,并克隆入pET-32a(+)中,在大肠杆菌BL21(DE3)中用IPTG诱导表达reBD-1融合蛋白。表达的融合蛋白扩大培养,进一步纯化后进行体外抑菌试验。结果表明,reBD-1前原肽和成熟肽扩增产物大小分别为215和138bp,目的基因的序列与驯鹿防御素-1mRNA序列同源性为100%。前原肽和成熟肽融合蛋白分子量分别为28和24ku。利用琼脂糖扩散法表明,0.08mg.mL-1的纯化成熟肽蛋白对金黄色葡萄球菌及大肠杆菌的活性有明显抵抗作用。结果显示,reBD-1前原肽及成熟肽在大肠杆菌中得到了高效表达,其成熟肽对革兰氏阳性菌和阴性菌的活性有抗性。

The aim of this experiment was to construct prokaryotic expression vector of reindeer β-defensin-1（reBD-1）,induce the expression of reBD-1 fusion protein in E.coli,and evaluate the bioactivity of the expression products.The prepro-peptide of reBD-1 was amplified by RT-PCR.The mature peptide of encoding reBD-1 was amplified from recombinant cloning vector PMD19T/reBD-1,and then cloned into pET-32a（＋）,in which reBD-1 fusion protein expression was induced by IPTG in E.coli BL21（DE3）.The expressed product was further cultured and purified for experiment of bacteriostasis in vitro.The results showed that the amplified products of prepro-peptide and mature peptide were 215 and 138 bp,respectively,and the homology of the sequences of the targeted gene and reBD-1 mRNA was up to 100%.The molecular weight of fusion proteins of prepro-peptide and mature peptide were 28 and 24 ku.The agar diffusion method has demonstrated that 0.08 mg·mL-1 purified mature peptide protein has obvious antimicrobial activity against S.aureus and E.coli.From the results we can conclude that the prepro-peptide and mature peptides have high expression level in E.coli,and the mature peptide has resistance to both Gram negative and Gram positive bacteria.