米曲霉giF-10内切葡聚糖酶基因在大肠杆菌中的表达

Expression of Aspergillus oryzae giF-10 Endoglucanase Gene in Escherichia coli

【目的】克隆米曲霉giF-10内切葡聚糖酶基因并检测其在大肠杆菌中的表达。【方法】以自行分离筛选出的天然米曲霉giF-10的基因组DNA为模板,PCR高保真扩增,扩增产物连接到pMD19-T克隆载体上,并构建原核表达载体,转化大肠杆菌BL21(DE3)。【结果】序列分析表明,其长度为1 251bp,编码417个氨基酸,序列比对发现与内切葡聚糖酶基因CelB序列相似性高达100%,将此基因注册GenBank(HQ739052)。刚果红水解圈筛选结果表明已成功表达。【结论】克隆了米曲霉giF-10内切葡聚糖酶基因,并在大肠杆菌成功表达,为纤维素酶工业化生产奠定基础。

[Objective] The aim of this research was to clone the gene encoding endoglucanase from Aspergillus oryzae giF-10 and detect the expression of the gene in Escherichia coli BL21 （DE3）. [Method] The gene encoding endoglucanase was cloned by PCR from Aspergillus oryzae giF-10, and inserted into the expression vector pET32a to express by IPTG induction in Escherichia coli. [Results] The gene was 1251 bp, encoding 417 amino acids, and was registered in GenBank （Ac- cession number HQ739052）. It shared a 100% sequence identity with the endoglucanase CelB gene by sequence alignment. The expression construct was transferred into Escherichia coli BL21 （DE3） and expressed successfully through hydrolysis cycle test. [Conclusion] The gene encoding endoglucanase was cloned from Aspergillus oryzae giF-10 and expressed successfully in Esche- richia coli BL21 （DE3）, which is important for industrial production of cellulase.