鸡β-防御素在大肠杆菌中的高效表达及复性  

Regulation of the expression of gallinacin-3 in Escherichia coli and refolding of the recombinant protein

作　　者：邢海云[1] 高英杰[2] 宁官保[3] 赵建增 乔彦良[1] 李朝阳[1] 

机构地区：[1]山东信得药业有限责任公司,山东青岛266071 [2]吉林大学,吉林长春130062 [3]山西农业大学动物科技学院,山西太谷030801

出　　处：《中国兽医学报》2007年第2期184-187,共4页Chinese Journal of Veterinary Science

基　　金：山东省科技发展计划资助项目(031030101)

摘　　要：鸡β-防御素(gallinacin-3,Gal3)是鸡体内重要的防御因子,本试验研究了鸡Gal3融合蛋白在大肠杆菌中高效表达的规律,并探索了重组蛋白质的复性工艺.结果表明,加入诱导剂后1h,开始有可检出量的重组蛋白质表达,2h的表达水平已接近最大量;化学诱导剂IPTG 0.2～1.2mol/L对重组蛋白的产量没有明显影响.经Lablmage软件分析,融合蛋白的产量可达菌体总蛋白的35%以上,且主要以包涵体形式存在.将所获包涵体用8mol/L尿素溶解后,分步稀释于含有氧化型谷胱甘肽和还原型谷胱甘肽的复性系统中,重组蛋白得到完全复性.12mg/L复性的重组鸡Gal3能抑制肠炎沙门氏菌的生长,24mg/L能抑制肠致病型大肠杆菌的生长.Defensins are a group of endogenous antimicrobial peptides that play indispensable function in triggering adaptive immune responses.Previously,we reported that the recombinant gallinacin-3,a chicken β-defensin,possess the strong antibacterial activities.In this paper,to investigate the possibility to produce the antimicrobial peptides in large scale for pharmaceutical purpose,procedures of the expression induction by Escherichia coli and renaturation of the peptide were studies.One hour since the induction with isopropylthio-β-D-galactoside(IPTG) detectable expression started and it reached maximal level at about 2 hours induction.Between 0.2-1.2 mol/L,there was no difference of IPTG concentration on the expression level.Most of the recombinant Gal-3 was in insoluble form,which making up around 35% of total bacteria proteins.Gal3-GST dissolving in 8 mol/L urea was progressively diluted into renaturation buffer containing glutathion and reduced glutathion and successfully regained its innate activities.It could completely depress Salmonella enteritidis and enterotoxigenic Escherichia coli in 12 mg/L and 24 mg/L,respectively.

关 键 词：鸡β-防御素 大肠杆菌 融合蛋白 抑菌作用 

分 类 号：S852.612[农业科学—基础兽医学]