转β-甘露聚糖酶基因大肠杆菌在猪肠道内的外泌型表达  

External Secretion of β-Mannase by Transgenic E.coli in Intestine of Swines

作　　者：王和平[1] 王龙[1] 文静[1] 高宏斌[1] 范文斌[1] 

机构地区：[1]内蒙古农业大学生物工程学院,呼和浩特010018

出　　处：《内蒙古大学学报（自然科学版）》2006年第1期58-64,共7页Journal of Inner Mongolia University：Natural Science Edition

基　　金：国家自然科学基金项目(39870558)

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.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 EcoR Ⅰ and BamH Ⅰ restriction sites to construct pGEM-Man Ⅰ . Sequencing of pGEM-Man I shows it＇s completely correct compared with the sequence of β-Mannase in Genbank. pGEM-Man Ⅰ was then digested by EcoR Ⅰ ,mungbean nuclease and Hind if ,and inserted into expression vector pT002 which contains Ω sequence,T7 promoter and signal peptide OmpT gene,to get the pT002-Man Ⅰ . 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-Man Ⅰ and pUC18-kil to get the external secreation system BL21-pT002-Man Ⅰ/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.

关 键 词：外分泌型表达 Β-甘露聚糖酶 RT-PCR 

分 类 号：Q786[生物学—分子生物学]