摘要
将PCR扩增得到的瑞氏木霉纤维素内切酶Ⅲ(Endo--β1,4-glucanaseⅢ,EGⅢ)基因片段插入巴斯德毕赤酵母(Pichia pastoris)质粒pPIC9K中,构建了EGⅢ的分泌表达质粒pPIC9K-eg3,然后经线性化后电转化导入P.pastorisGS115受体菌中。获得的阳性克隆经SDS-PAGE和酶活检测,证明工程菌株发酵上清液中含有表达的EGⅢ蛋白并具有纤维素内切酶酶活。通过荧光定量PCR确定了各菌株的拷贝数并对不同拷贝数范围的菌体生长和EGⅢ的表达做了比较。实验结果表明,低拷贝重组菌有着相对较好的EGⅢ表达量。
The endo-beta-1,4-glucanase （EGⅢ） gene （EGⅢ） was amplified from fungus Trichoderma reesei using PCR thehnique. The EGⅢ was inserted into pPIC9K, a Pichia paswris vector, to yield the recombinant secrete plasmid pPIC9K-eg3. The pPIC9K-eg3 was transformed into P. pastoris GS115. The EGⅢ was expressed and secreted into the broth. The insert genels copy number in host genomic DNA were detected by FQ-PCR. The cell-growth and EGⅢ activity of recombinant with different copies of inserted EGⅢ were analyzed. The resalts suggest that the strain with single copy showed better EGⅢ expressis than the strains with higher gene copy numbers.
出处
《生物加工过程》
CAS
CSCD
2006年第4期12-16,共5页
Chinese Journal of Bioprocess Engineering
基金
国家973项目(No.2003CB716000)
国家自然科学基金项目(No.30370036)
山东大学微生物技术国家重点实验室开放基金项目
