斜卧青霉L-06内切葡聚糖酶Ⅰ基因的克隆与表达

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

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。

Abstract:

[Objective] The research focus on cloning endoglucanase I (egI) gene from Penicillium 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 constructing prokaryotic expression system and expressing egI gene.