【作者】 张金波；

【导师】 陈创夫； 祝建波；

【作者基本信息】 石河子大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 ETEC肠毒素分为不耐热性肠毒素(heat-labile,LT)和耐热性肠毒素(heat-stable,ST)两种,LT是由A、B两种亚单位组成的AB5型结构的六聚体蛋白,是目前人们发现的最强有力的黏膜免疫原和黏膜免疫佐剂之一。其中A亚单位具有ADP核糖基化酶活性, A亚单位通过其A2片段插入B亚单位五聚体“戒孔”中央,组成一个完整的LT分子。LT-B可与小肠黏膜上皮细胞GM1神经节苷脂受体结合,导致有毒性的A亚单位内在化和吸收,从而诱发全身和局部抗体反应。融合保护性抗原的LT类全毒素具有传输抗原载体和免疫佐剂两重功效。本研究利用基因工程技术,首先克隆热稳定性肠毒素STII的抗原决定簇(CEHYRQIAKESCKKGF)与K88ac蛋白融合基因,并利用套叠PCR技术定点突变K88ac-STII内部EcoR I、Sac I酶切位点。将LT的A亚基的LTA1部分用以已克隆的抗原基因K88ac-STII替换,并将烟草病程相关蛋白PR1a基因的信号肽分别与K88ac-STII-LTA2、LTB连接,构建到pCAMBIA2300植物高效表达载体上,即构建了带有信号肽的PR1as-K88ac-STII-LTA2 /Ubquitin-PR1as-LTB双价植物表达载体。构建的重组质粒分别进行PCR检测、酶切分析及核苷酸序列分析,结果表明,插入的融合基因核苷酸序列与预期的一致,且阅读框正确。重组质粒通过冻融法转化根癌农杆菌GV3101,利用农杆菌介导的叶盘法转化模式植物烟草。转基因烟草经PCR和RT-PCR初步检测,证明外源基因已经整合到受体植物基因组中,同时表明融合基因在转录水平上有表达。提取转基因烟草植株总蛋白质, Western-dot免疫检测验证了转基因植株表达的重组抗原有免疫原性。本研究结果表明,K88-STII-LTA2/LTB融合基因能够在烟草中正确表达并证实该基因具有一定的免疫原性,转基因烟草的获得为下一步把LT类全毒素转入苜蓿等饲料作物进行转基因抗腹泻植物口服疫苗的研究奠定了良好的基础。更多还原

【Abstract】 Enterotoxigenic Escherichia coli (ETEC) included heat-labile enterotoxin and heat-stable enterotoxin . Heat-labile enterotoxin (LT) was an AB5-typed mixed six-polyprotein,which was one of the strongest immunogenicity and mucosal immunoadjuvanticity. A subunit has the ADP ribosylation enzyme activity. A subunit through its A2 fragment inserted the hole of B subunit five-polyprotein, which composed a complete LT.LT-B had the ability of combining with the small intestine mucous epithelium ganglioside GM1 acceptor, which caused the virulent A subunit intermality and absorption, thus induced the whole body and the partial immune antibody response.LT holotoxin-like fused protective antigen have two functions of carrier of delivery antigen and immunoadjuvant.In this reach, with gene engineering technology the fusion gene which was antigenic determinant (CEHYRQIAKESCKKGF)of heat-stable toxin STII and K88ac protein was cloned. The fusion gene EcoR I、Sac I sites were site-specific mutated by overlap extension of PCR technique.The high performance two-gene plant expression vector Pr1as-K88ac-STII-LTA2/UP-LTB was constructed,which included ubiquitin and signal peptide sequence of pathogenesis-related protein 1a (PR1a). Recombinant plasmids were amplified by PCR, restriction endonuclease analysis and DAN sequencing. The results indicated that the recombinant plasmids were correctly constructed. The recombinant plasmid was transferred into Agrobacterium tumefaciens GV3101 respectively using the freezethaw method. Transgenic tobacco plants recovered from leaf discs applying Agrobacterium mediated gene were selected on solid medium containing Kanamycin. They were detected by PCR analysis, the results showed that the foreign gene have been integrated into tobacco genome.RT-PCR indicated the fusion gene of transgenic plantlet has expressed at the transcriptional level. Based on Western dotting, results showed that recombinant protein expressed by transgenic tobacco plants could eliciting immunoresponse to LTB and LT antibody.The results proved that the fused genes K88-STII-LTA2/LTB could expressed correctly in transgenic tobacco and had immunogenity. The study will lay a foundation for further study of new plant edible vaccines to diarrhea in alfalfa.更多还原