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External Secretion of β-Mannase by Transgenic E.coli in Intestine of Swines
Author(s): 
Pages: 58-64
Year: Issue:  1
Journal: ACTA SCIENTIARUM NATURALIUM UNIVERSITATIS NEIMONGOL

Keyword:  外分泌型表达β-甘露聚糖酶RT-PCR;
Abstract: 从里氏木霉Rut C-30提取总RNA,用RT-PCR方法克隆到β-甘露聚糖酶成熟肽cDNA,并将其EcoRΙ和BamH I酶切后连接到克隆载体pGEM进行测序,结果与GenBank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-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,构建成表达载体pTOO2-ManΙ.然后利用大肠杆菌素释放基因(kil)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pTOO2-ManΙ和质粒pUC18-kil共转化到大肠杆菌BL21株中,构建成外泌型表达体系BL21-pTOO2-ManΙ/pUC18-kil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ELISA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.
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