松材线虫纤维素酶基因的克隆及其在大肠杆菌中的表达、纯化和性质研究
摘要
采用PCR技术扩增了松材线虫纤维素酶的编码基因,克隆基因与已报道的纤维素酶BXC10基因的同源性高达99%。将其克隆到组成型表达载体pET-15b上构建pET-15b-BXC,并将该重组质粒转化大肠杆菌BL21(DE3)菌株构建工程菌,SDS-PAGE分析表明,IPTG诱导了纤维素酶在工程菌中表达。采用Ni2+-NTA树脂和阴离子(DEAE)交换树脂对重组蛋白分离纯化,SDS-聚丙烯酰胺凝胶电泳检测其纯化程度,DNS法测定其纤维素酶的活性及其最适温度、最适pH和其热稳定性、pH稳定性。
SDS-聚丙烯酰胺凝胶电泳分析表明,亲和层析后重组蛋白质的纯度为50%左右,经阴离子(DEAE)交换树脂分离纯化后,只得到均一的一条带,大小为40 kDa。活性分析表明工程菌表达的重组蛋白具有纤维素酶活性,其最适温度为45℃,最适pH为40。实验结果还证明该蛋白具有很好的热稳定性：不同温度处理1h后,该蛋白在45℃-65℃范围内可保持70%以上的纤维素酶酶活力,70℃以后,其纤维素酶酶活力下降迅速,80℃时其纤维素酶酶活力几乎完全丧失,45℃时剩余纤维素酶酶活最高,可达98%；该蛋白pH稳定性也很好：其纤维素酶酶活在pH3.0-5.0范围内其酶活稳定性逐步上升,并在pH5.0时酶活稳定性达到最高。本实验结果可为进一步研究松材线虫纤维素酶在松萎蔫病中的作用奠定基础。
A gene encoding cellulase from Bursaphelenchus xylophilus was cloned by PCR, and showed 99% homology to the reported gene of cellulase BXC10. The gene was cloned into pET-15b to construct expressing vector pET-15b-BXC. Then, this recombinant plasmid was introduced into E.coli BL21(DE3) to construct engineering bacterium. Recombinant cellulase was successfully expressed in engineering bacterium induced by IPTG as analyzed by SDS-PAGE. Recombinant protein was purified by Ni2+-NTA resin and anionic (DEAE) exchange resin, its purification degree was assayed by SDS-PAGE, its cellulase activity was determined by DNS method, and its optimum temperature、the optimal pH、thermal stability and pH stability was also determined by DNS method.
The results of SDS-PAGE showed that the purity of recombinant protein was approximate 50% by affinity chromatography, and there was only a uniform band of 40 kDa after purified by anionic (DEAE) exchange resin. The results determined by DNS method showed that recombinant protein expressed by engineering bacteria had good cellulose enzyme activity, the optimal temperature and pH of the recombinant cellulase were 45℃and 4.0, respectively. Experimental results proved that this protein had very good thermal stability. After treated under different temperatures for 1 h, the protein could keep more than 70 percent of the cellulase enzyme activity in a range between 45℃to 65℃, while its cellulase enzyme activity declined rapidly over 70℃. It almost lost cellulase enzyme activity completely when the temperature reached 80℃, and the rest cellulase enzyme activity of the protein was highest at 45℃, up to 98%. The pH stability of this protein was also very good. Its stability rose gradually in a range of pH3.0 to pH5.0, and the left activity was highest at pH5.0. The results can lay the foundation for further research about the role of cellulase in pine wilt disease.
SDS-聚丙烯酰胺凝胶电泳分析表明,亲和层析后重组蛋白质的纯度为50%左右,经阴离子(DEAE)交换树脂分离纯化后,只得到均一的一条带,大小为40 kDa。活性分析表明工程菌表达的重组蛋白具有纤维素酶活性,其最适温度为45℃,最适pH为40。实验结果还证明该蛋白具有很好的热稳定性：不同温度处理1h后,该蛋白在45℃-65℃范围内可保持70%以上的纤维素酶酶活力,70℃以后,其纤维素酶酶活力下降迅速,80℃时其纤维素酶酶活力几乎完全丧失,45℃时剩余纤维素酶酶活最高,可达98%；该蛋白pH稳定性也很好：其纤维素酶酶活在pH3.0-5.0范围内其酶活稳定性逐步上升,并在pH5.0时酶活稳定性达到最高。本实验结果可为进一步研究松材线虫纤维素酶在松萎蔫病中的作用奠定基础。
A gene encoding cellulase from Bursaphelenchus xylophilus was cloned by PCR, and showed 99% homology to the reported gene of cellulase BXC10. The gene was cloned into pET-15b to construct expressing vector pET-15b-BXC. Then, this recombinant plasmid was introduced into E.coli BL21(DE3) to construct engineering bacterium. Recombinant cellulase was successfully expressed in engineering bacterium induced by IPTG as analyzed by SDS-PAGE. Recombinant protein was purified by Ni2+-NTA resin and anionic (DEAE) exchange resin, its purification degree was assayed by SDS-PAGE, its cellulase activity was determined by DNS method, and its optimum temperature、the optimal pH、thermal stability and pH stability was also determined by DNS method.
The results of SDS-PAGE showed that the purity of recombinant protein was approximate 50% by affinity chromatography, and there was only a uniform band of 40 kDa after purified by anionic (DEAE) exchange resin. The results determined by DNS method showed that recombinant protein expressed by engineering bacteria had good cellulose enzyme activity, the optimal temperature and pH of the recombinant cellulase were 45℃and 4.0, respectively. Experimental results proved that this protein had very good thermal stability. After treated under different temperatures for 1 h, the protein could keep more than 70 percent of the cellulase enzyme activity in a range between 45℃to 65℃, while its cellulase enzyme activity declined rapidly over 70℃. It almost lost cellulase enzyme activity completely when the temperature reached 80℃, and the rest cellulase enzyme activity of the protein was highest at 45℃, up to 98%. The pH stability of this protein was also very good. Its stability rose gradually in a range of pH3.0 to pH5.0, and the left activity was highest at pH5.0. The results can lay the foundation for further research about the role of cellulase in pine wilt disease.
引文
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