香蕉内生拮抗细菌KKWB-5的几丁质酶在大肠杆菌中的表达纯化与复性研究  

The Study on Renaturation,Expression and Purification of Chitinase from Endophytic Bacterium KKWB-5 Against Banana Fusarium Wilt in Escherichia coli

作　　者：夏启玉[1] 顾文亮[2] 卢娟 张欣 卢雪花[1] 王宇光[1] 

机构地区：[1]中国热带农业科学院热带生物技术研究所农业部热带作物生物技术重点开放实验室,海口571101 [2]中国热带农业科学院香料饮料研究所,海南万宁571533 [3]广西壮族自治区玉林市林业科学研究所,广西玉林537501 [4]中国热带农业科学院环境与植物保护研究所,海南儋州571737

出　　处：《中国农学通报》2013年第33期307-312,共6页Chinese Agricultural Science Bulletin

基　　金：中央级公益性科研院所基本科研业务费专项"香蕉枯萎病生防菌的筛选和综合防治措施的研究"(ITBBZX2008-2-3)

摘　　要：将香蕉内生拮抗细菌KKWB-5的几丁质酶在大肠杆菌中的表达,旨在检测该酶对香蕉枯萎病病原菌4号小种的抗性。PCR扩增该基因连入大肠杆菌表达载体pET22b,导入大肠杆菌BL21(DE3),IPTG诱导重组菌株,SDS-PAGE分析目的蛋白的表达,对目的蛋白进行纯化,并将纯化后的目的蛋白复性,对复性产物进行水解几丁质活性和拮抗香蕉枯萎病活性的分析。SDS-PAGE分析表明目的蛋白在大肠杆菌中以包涵体的形式大量表达,经变性Ni2+柱纯化得到了高纯度的重组蛋白,表达量达293 mg/L;稀释和透析2种复性方法中,透析复性法的目的蛋白得率较高,为84.6%。复性后的目的蛋白具有水解几丁质的活性,同时对香蕉枯萎病病原菌也具有一定的抗性,但抗性较弱。该几丁质酶在大肠杆菌中得到高效表达,且复性后的几丁质酶对香蕉枯萎病病原菌4号小种有一定的抑制作用,该酶对KKWB-5菌株拮抗香蕉枯萎病菌的能力有一定的贡献。The chitinase from endophytic bacterium KKWB-5 was expressed in Escherichia coli to identify the antagonistic activity of the chitinase against Fusarium oxysporum f.sp cubense race 4（Foc4）. The chitinase gene amplified by PCR was inserted into the expression vector pET22b,and the recombinant plasmid was transformed into E. coli BL21（DE3）. The recombinant strain was induced with IPTG,and SDS-PAGE was used to analyze the expression of objective protein in E. coli. Then the recombinant protein was purified and renatured,and the hydrolysis activity and the antagonistic activity against Foc4 of the renatured protein were determined. The results of SDS-PAGE analysis showed that the recombinant objective protein was abundantlyexpressed in the form of inclusion body in E. coli. Recombinant protein with high purity was purified from inclusion body protein by denaturated Ni2 ＋affinity chromatography. The yield of objective protein reached 293 mg/L culture. The yields of refolding protein using dialysis renaturation which was 84.6%,was higher than that using dilution renaturation. The activities tests indicated that the renatured protein was hydrolytic of chitin,and was antagonistic against Foc4,but the antagonistic activity was weak. In conclusion,the chitinase from endophytic bacterium KKWB-5 was effectively expressed in E. coli,and the renatured chitinase was resistant to Foc4 to some extent. Thus the chitinase made some contributions to the antagonistic activity against Foc4 ofendophytic bacterium KKWB-5.

关 键 词：拮抗细菌 几丁质酶 表达纯化 复性 活性分析 

分 类 号：Q786[生物学—分子生物学]