摘要
侧孢短芽孢杆菌G4菌株在侵染线虫的过程中可以分泌蛋白酶,降解线虫体壁,从而帮助细菌侵入宿主体内.在本文中,侧孢短芽孢杆菌的胞外蛋白酶BLG4基因经克隆后插入到改造后的枯草芽孢杆菌表达载体pWT22中,构建重组表达质粒pWT22-BLG4.构建成功的重组质粒通过化学转化法转入枯草芽孢杆菌蛋白酶缺失菌株WB600中,转化子pWT22-BLG4/WB600在IPTG(1.0 mmol.L-1)的诱导下,发酵液上清中的蛋白酶活可达到620 U.mL-1,高于出发菌株G4的BLG4酶活(240 U.mL-1).经SDS-PAGE分析,重组子pWT22-BLG4/WB600产生了一条分子质量约为30 ku的条带,该条带的大小与侧孢短芽孢杆菌G4上清发酵液中BLG4条带的大小一致.而未经IPTG诱导的重组子pWT22-BLG4和经IPTG诱导的WB600则未出现该条带.结果证明侧孢短芽孢杆菌G4的碱性蛋白酶基因BLG4已在枯草芽孢杆菌WB600中获得了成功表达.重组蛋白酶具有与侧孢短芽孢杆菌G4产生的BLG4蛋白酶同样的酶学性质.同时,重组蛋白酶具有明显的杀线虫和体壁降解能力,表明其在线虫生物防治中将有广泛的应用前景.
Protease BLG4 from Brevibacillus laterosporus G4 has been proposed as an important virulence factor in bacterial pathoge nicity against nematodes. In this paper, alkaline protease gene BLG4 from B. laterospoms G4 was cloned and inserted into an expres sion vector pWT22 to construct a recombinanted plasmid pWq22 BLC,4. The pWT22 BLG4 was transformed into Bacill~ subtils WB600 by chemical method. After IPTG （ 1.0 mmol·L^-1 ） induction, the protease activity of the fermentation supematant of the transformant pWT22 BLG4/WB600 reached 620 U· mL^-l, which was higher than native BLG4 enzyme activity produced by the wild strain G4. SDS PAGE analysis revealed that the transformant with recombinanted expression plasmid pWT22 BLG4 had an ad ditional 30 ku protein band, which was identical to B. laterosporus G4. However, the additional band was not present in the negative control Bacillus subtilis WB600. There were no significant differences between the recombinanted and native BLG4 with regard to en zymatic properties. In further bioassays, the reeombinanted protease BLG4 had significant nematotoxic activity and ability to degrade nematode cuticles, which indicated that it had a potential as a bio control agent of plant parasitic nematodes in the future..
出处
《福建农林大学学报（自然科学版）》
CSCD
北大核心
2011年第2期165-171,共7页
Journal of Fujian Agriculture and Forestry University:Natural Science Edition
基金
国家自然科学基金项目(30800735)
福建省自然科学基金(2009J05057
2008F3036)资助
关键词
侧孢短芽孢杆菌
侵染性蛋白酶
枯草芽孢杆菌
高效表达
酶学特性
线虫
Brevibacillus laterosporus
infectious protease
Bacillus subtilis
over expression
enzymatic property
nematode
