【作者】 文静；

【导师】 王和平；

【作者基本信息】 内蒙古农业大学 ， 基础兽医学， 2004， 硕士

【摘要】 本研究将含有β-甘露聚糖酶基因(ManI)的重组质粒pGEM-ManΙ经EcoRΙ、绿豆芽核酸酶、HindⅢ酶切处理和纯化后，获得目的基因（β-甘露聚糖酶基因）。同时，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pT002经BamHΙ、绿豆芽核酸酶、HindⅢ酶切处理。将目的基因连接在pT002质粒信号肽OmpT之后，构建表达载体pT002-ManΙ。将表达载体pT002-ManΙ转化到大肠杆菌BL21（DE3）感受态细胞中，通过筛选获得阳性克隆子。对阳性克隆子进行PCR及酶切鉴定，并对载体中插入的目的基因进行测序。测序结果与GenBank中的里氏木霉RutC-30的β-甘露聚糖酶成熟肽cDNA序列同源性比较，结果二者序列完全一致。将表达载体pT002-ManΙ和质粒pUC18-kil共转化到大肠杆菌BL21（DE3）株，构建了外分泌型表达体系，经IPTG诱导，重组蛋白获得高效表达，SDS-聚丙烯酰胺凝胶电泳分析表明，表达的重组蛋白分子量约为51kD。将培养液浓缩纯化后，以槐豆胶为底物用3,5—二硝基水杨酸法（DNS）测定培养液中β-甘露聚糖酶的活力。纯化后的产物具有明显的β-甘露聚糖酶活性，证明重组β-甘露聚糖酶在分泌过程中得到正确加工。更多还原

【Abstract】 In this study, the target DNA fragment of ManI gene was purified from the recombinant plasmid pGEM-ManΙ after digesting by restriction enzyme EcoRI Mung Bean Nuclease、HindⅢ, and then cloned into secretion expression vector pT002 containing Ω sequence, T7 promoter and signature sequence OmpT after digesting by restriction enzyme BamHΙ、Mung Bean Nuclease、HindⅢ. The ManI gene was fused in-frame after the signature sequence ompT in the resulting plasmid pT002-ManΙ. The positive clones were screened in E.coli BL21 transformed by the recombinant plasmid and then subjected to sequencing after identification by PCR. The sequence of cloned ManI gene are completely identical to corresponding cDNA sequences of Trichoderma Reesei Rut- C30 of β-Mannanase mature peptides. The secretion expression system was established with E.coli BL21 co-transformed by pT002-ManΙ and pUC18-kil according to the principle kil can enhance the permeabilization of cell membrane and promote the secretion of periplasmic protein. The fusion β-Mannanase with molecular weights of about 51 kD as indicated by SDS-PAGE analysis was expressed with high-efficency after inducing by IPTG. The activity of β-Mannanasein in the concentrated medium was determined with locost bean gum as substract by 3,5-dinitosalicylic acid method, and the result indicated that the purified product showed apparentβ-Mannanase activity and the recombinant β-Mannanase was correctly processed during the secretion.更多还原