【摘要】 从里氏木霉R u t C-30提取总RNA,用RT-PCR方法克隆到β-甘露聚糖酶成熟肽cD-NA,并将其E coRΙ和B amH I酶切后连接到克隆载体pGEM进行测序,结果与G enB ank公布的完全一致,随后将含有β-甘露聚糖酶基因的重组质粒pGEM-M anΙ经E coRΙ、绿豆芽核酸酶、H indⅢ酶切处理和纯化后,与含有Ω序列和T 7启动子以及信号肽Om pT序列的分泌型表达载体pTOO 2连接,构建成表达载体pTOO 2-M an.Ι然后利用大肠杆菌素释放基因(k il)能有效的增加细菌外膜通透性促进周质蛋白外泌的原理,再将表达载体pTOO 2-M anΙ和质粒pUC 18-k il共转化到大肠杆菌BL 21株中,构建成外泌型表达体系BL 21-pTOO 2-M anΙ/pUC 18-k il进行外泌型表达.表达产物经酶活鉴定具有明显的β-甘露聚糖酶活性,经EL ISA双抗夹心法检测,无论在体外或猪肠道内容物内均为阳性,从而实现了该基因的外泌型表达;表达产物经SDS-聚丙烯酰胺凝胶电泳鉴定,分子量为50.98 kD和53.98 kD,该工程菌通过瘘管灌注到猪肠道内进行表达研究,结果基本令人满意.从而实现了该转基因大肠杆菌在猪肠道中外泌型表达的目的.更多还原

【Abstract】 The cDNA of β-Mannase mature peptide was cloned by RT-PCR from total RNA of Trichoderma Reesei-Rut30,then ligated into vector pGEM by EcoRⅠand BamHⅠ restriction sites to construct pGEM-ManⅠ.Sequencing of pGEM-ManⅠshows it′s completely correct compared with the sequence of β-Mannase in Genbank.pGEM-ManⅠwas then digested by EcoRⅠ,mungbean nuclease and HindⅢ,and inserted into expression vector pT002 which contains Ω sequence,T7 promoter and signal peptide OmpT gene,to get the pT002-ManⅠ.It is well known that gene product kil released by colicin could increase the permeability of E.coli outer membrane and promote secretion of periplasmic protein.We co-transformed BL21 with both pT002-ManⅠ and pUC18-kil to get the external secreation system BL21-pT002-ManⅠ/pUC18-kil.The system was put into swine intestine by fistula perfusion and the expressed product was extracted.Enzyme activity assay of the expression prodult proved the β-Mannase activity.Double antibody assay also showed positive both in vitro and in swine intestine.SDS-PAGE showed the expression product has molecular weight of 50.9 kD and 53.98 kD respectively.更多还原