含有MAR序列的STI-STII-LTB融合基因在苜蓿中的表达

Expression of the fusion gene STI-STII-LTB flanked by martix attachment region sequence MARs in alfalfa

产肠毒素性大肠杆菌(ETEC)LT和ST是导致人和动物腹泻的主要病原菌。利用三引物PCR法和酶切实现了STI、STII、LTB基因的融合,LTB基因的5′位于STII基因的3′端,三者在同一阅读框。将融合基因STI-STII-LTB构建到带有核基质结合区序列(MARs)的植物表达载体pBI121-MARs中GUS基因的位置。同时构建不含MARs序列的重组质粒。重组质粒pBI121-MARs-STI-STII-LTB、pBI121-STI-STII-LTB通过冻融法转化根癌农杆菌,并通过农杆菌介导法转化杂花苜蓿。转基因苜蓿植株经PCR检测、Southern-blotting分析表明,转基因苜蓿植株基因组中可检测到STI-STII-LTB融合基因;提取转基因苜蓿植株总蛋白质,SDS-PAGE结果显示,转基因植株表达了重组抗原蛋白,且含MAR序列的蛋白表达量要高于不含MAR序列的表达量;Western-dotting免疫检测证实转基因植株表达的重组抗原具有免疫原性。

Enterotoxigenic Escherichia coli(ETEC)are responsible for many diarrheal syndromes.The genes encoding heat-stable toxinⅠandⅡ(STI,STII),heat-labile toxin B subunit of ETEC were fused by TP-PCR and restriction en- donuclease.The 5 terminus of the gene encoding LTB was genetically fused to the 3 terminus of the ST gene.The fused gene STI-STII-LTB was cloned into plant expression vector pBI121-MARs to replace GUS gene,another plant expression vector was constructed without MARs.The recombinant plasmid pBI121-...