鸡β-防御素-1基因(Gal-1)在大肠杆菌中的融合表达与纯化

The fusion expression and purification of chicken β-defensin-1(Gal-1)gene in Escherichia coli

内源性抗菌多肽防御素是机体先天性免疫系统的重要组成部分,可帮助机体防御外界病原微生物的入侵。鸡β-防御素-1(G a llinac in-1,G a l-1)对许多致病菌都具有杀菌作用,具有很好的研究开发价值。本研究利用PCR技术从重组质粒pGEM-TE asy-ga l-1中克隆出G a l-1基因成熟肽区,将该基因插入原核表达载体pET-32a(+)中,构建重组质粒pET-32a(+)-ga l-1,然后用重组质粒转化大肠杆菌BL-21。挑选阳性克隆菌,用浓度为0.5 mm o l/L的IPTG进行诱导表达。SDS-PAGE电泳显示在目标位置约25kD处出现了条带并用W erstern B lot进行了鉴定。表明G a l-1基因在大肠杆菌以融合形式得到了表达。经灰度扫描显示重组蛋白表达量占细菌总蛋白的15.54%。用50%N i-NTA纯化树脂进行层析纯化,在洗脱液E中出现单一条带。G a l-1多肽在pET-32a(+)质粒表达体系中成功表达为鸡防御素多肽下一步的研究奠定基础。

Endogenous antimicrobial peptide defensins are essential components of innate immunity. Defensins are distributed throughout the host to control invading micro-organisms. Chicken beta-defensin-1 （Gallinacin-1,Gal-1） has antimicrobial activity against potentially pathogens and is worthwhile being investigated and developed. The mature fragment of Gal-1 gene was amplified from the plasmid of pGEM-T Easy-gal-1, and then inserted into the EcoRⅠ and BamH Ⅰ enzyme-cutting sites of pET-32a （＋） plasmid. The recombinant vectors named pET-32a （＋）-gal-1 were transformed into the competent cell E. coli BL-21. The positive clones were cultured and induced to express target protein by addition of 0. 5 mmol IPTG in LA. Expression products with molecular weight of approximately 25 kDa were examined by SDS-PAGE and Western Blot, which showed that gal-1 gene was expressed in the form of fusion protein. Densitometric scanning analysis demonstrated that the fusion protein accounted for about 15.5% of the total bacterial protein. The expressed fusion proteins were purified with 50% Ni-NTA resin affinity chromatography. Highly purified proteins were achieved in the eluate. Further study will be continued based on the results of this research.