里氏木霉Rut-C30β-甘露聚糖酶基因表达载体的构建

THE RELEASE EXPRESSION VECTOR CONSTRUCTION OF β-MANNASE GENE OF TRICHODERMA REESEI-RUT C30 IN E.COLI

将含有β-甘露聚糖酶基因的重组质粒pGEM-ManΙ经EcoRΙ、绿豆芽核酸酶、HindⅢ酶切处理和纯化后,与含有Ω序列和T7启动子以及信号肽OmpT序列的分泌型表达载体pTOO2连接,将质粒pGEM-ManΙ中的β-甘露聚糖酶基因连接在pTOO2质粒信号肽OmpT之后,构建成表达载体pTOO2-ManΙ。将表达载体pTOO2-ManΙ转化到大肠杆菌BL21(DE3)感受态细胞中,克隆转化子,pCR测序,结果获得了β-甘露聚糖酶编码的成熟肽cDNA,其序列与GeneBank报道完全一样。并从该克隆转化的大肠杆菌的周质中检测到了β-甘露聚糖酶的活性同时,证明β-甘露聚糖酶基因在分泌型过程中得到正确加工。

After cutted by restriction enzyme and purified β-Mannase gene of recombinant plasmid p^(GEM)-ManΙwas inserted into expression p^(T002) with Ω sequence that was ligated in front of a T7 promoter and OmpT signal sequence. E.coli BL21(DE3) was cotransformed with p^(TOO2)-ManΙ,cloned transformant,determined the sequence after PCR?A mature peptide cDNA coding β-Mannanase gene was acquired,and the cDNA sequence was equal to completely one reported by Genbank?Theβ-Mannase product activity could be detected in E.coli BL21(DE3) with Theβ-Mannase gene ?