作　　者： ; ; ; ; ; ;

机构地区： 汕头大学理学院生物系

出　　处： 《微生物学通报》 2012年第5期696-701,共6页

摘　　要： 【目的】克隆斜卧青霉L-06的内切葡聚糖酶Ⅰ基因(egI),并实现其在大肠杆菌内的高效表达。【方法】利用RT-PCR技术克隆了斜卧青霉L-06的内切葡聚糖酶Ⅰ基因(egI),并将egI基因克隆到原核表达载体中,构建了重组质粒pET32a-egI。【结果】转化至大肠埃希菌Rosetta(DE3),经IPTG诱导重组蛋白表达,SDS-PAGE检测结果表明:重组表达产物的相对分子质量约为80 kD,与预期相符。重组表达的菌悬液,经破碎离心,取其上清液,进行纤维素酶活性染色,获得了活性条带。DNS法测得内切酶活力为2.56 IU/mL。【结论】构建了斜卧青霉L-06内切葡聚糖酶Ⅰ的原核表达系统。 [Objective] The research focus on cloning endoglucanase I（egI） gene from Peni-cillium decumbens L-06 and expressing in Escherichia coli with high efficiency.[Methods] egI gene was cloned from Penicillium decumbens L-06 by RT-PCR method.Recombinant plasmid pET32a-egI was constructed and was transformed into Escherichia coli rosetta（DE3）.Recombinant protein with His-tag was expressed in E.coli rosetta（DE3） after induction with IPTG and then was purified with the Ni-NTA affinity chromatography.[Results] As expected,the relative molecular mass was approximately 80kD after analyzed by SDS-PAGE and Western blotting.Hydrolysis activity of recombinant protein was assayed by cellulase activity staining and DNS method（2.56 IU/mL）.[Conclusion] The results achieve the purpose as con-structing prokaryotic expression system and expressing egI gene.

关 键 词： 斜卧青霉 内切葡聚糖酶 克隆 原核表达

领　　域： [生物学]