CTB-PROIN和CTB-INSB融合基因构建及其在大肠杆菌和植物中的表达

发布时间：2018-01-27 16:07

  本文关键词： 霍乱毒素B亚单位 胰岛素原 胰岛素B链 大肠杆菌 转基因马铃薯　出处：《宁夏大学》2005年硕士论文　论文类型：学位论文

【摘要】：口服胰岛特异的自身抗原可诱导免疫耐受,防止自发性自身免疫糖尿病的发生。为使胰岛素蛋白更有效地被肠相关淋巴组织吸收,本研究分别构建了强免疫佐剂霍乱毒素B亚单位(cholera toxin B subunit, CTB)与胰岛素原(proinsulin, PROIN)和胰岛素B链(insulin B chian, INSB)的融合基因CTB-PROIN、CTB-INSB,探索了利用马铃薯生产1型糖尿病口服疫苗的可能性,主要研究内容和结果如下: 1.首先将融合基因CTB-PROIN、CTB-INSB克隆到大肠杆菌表达载体pET-30a(+)中,并诱导其表达。大肠杆菌表达的融合蛋白CTB-PROIN和CTB-INSB可与GM1糖苷脂结合,并具备CTB和胰岛素的双重抗原性。 2.在此基础上,分别构建了含有土豆块茎特异性启动子Patatin、融合基因、马铃薯蛋白酶抑制剂基因Ⅱ(Pin2)的3’末端非翻译区(UTR)序列和NOS终止子的两个植物转化载体,通过农杆菌介导法转化马铃薯,并获得含有融合基因的转基因马铃薯植株。转基因植物表达的融合蛋白具有霍乱毒素和胰岛素的双重抗原性,可于神经节苷脂GM1结合,具有潜在的生物活性。这为利用转基因马铃薯免疫NOD小鼠,进一步分析其免疫特性奠定了基础。
[Abstract]:Oral islet specific autoantigen can induce immune tolerance and prevent spontaneous autoimmune diabetes mellitus. In order to make insulin protein more effectively absorbed by intestinal associated lymphoid tissue. In this study, cholera toxin B subunits (CTBs) and proinsulin were constructed respectively. The fusion gene CTB-PROIN-CTB-INSB of proin and insulin B strand insulin B chian (INSBs). The possibility of using potato to produce oral vaccine for type 1 diabetes mellitus was explored. The main contents and results were as follows: 1. Firstly, the fusion gene CTB-PROINOCTB-INSB was cloned into E. coli expression vector pET-30a (). The fusion proteins CTB-PROIN and CTB-INSB expressed by Escherichia coli can bind to GM1 glycosides and possess the double antigenicity of CTB and insulin. 2. On this basis, the potato tuber specific promoter Patatin was constructed, and the fusion gene was constructed. Potato protease inhibitor gene 鈪，

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