Cloning and expression of egI gene from Penicillium decumbens L-06
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【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.