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A STABLE PLASTID TRANSFORMATION AND EXPRESSION VECTOR FOR STABLY TRANSFORMING A PLASTID GENOME

机译：稳定转化质体基因组的稳定质体转化和表达载体

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Transgenic chloroplast technology could provide a viable solution to the production of Insulin-like Growth Factor I (IGF-I), Human SerµmAlbumin (HSA), or interferons (IFN) because of hyper-expression capabilities, ability to fold and process eukaryotic proteins with disulfide bridges (thereby eliminating the need for expensive post-purification processing). Tobacco is an ideal choice because of its large biomass, ease of scale-up (million seeds per plant), genetic manipulation and impending need to explore alternate uses for this hazardous crop. Therefore, all three human proteins will be expressed as follows: a) Develop recombinant DNA vectors for enhanced expression via tobacco chloroplast genomes' b) generate transgenic plants c) characterize transgenic expression of proteins or fusion proteins using molecular and biochemical methods d) large scale purification of therapeutic proteins from transgenic tobacco and comparison of current purification / processing methods in E.coli or yeast e) Characterization and comparison of therapeutic proteins (yield, purity, functionality) produced in yeast or E.coli with transgenic tobacco f) animal testing and pre- clinical trials for effectiveness of the therapeutic proteins. Mass production of affordable vaccines can be achieved by genetically engineering plants to produce recombinant proteins that are candidate vaccine antigens. The B subunits of Enteroxigenic E. coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are examples of such antigens. When the native LTB gene was expressed via the tobacco nuclear genome, LTB accumulated at levels less than 0.01% of the total soluble leaf protein. Production of effective levels of LTB in plants, required extensive codon modification. Amplification of an unmodified CTB coding sequence in chloroplasts, up to 10,000 copies per cell, resulted in the accumulation of up to 4.1% of total soluble tobacco leaf protein as oligomers (about 410 fold higher expression levels than that of the unmodified LTB gene). PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that chloroplast synthesized CTB assembled into oligomers and was antigenically identical to purified native CTB. Also, GM1-rganglioside binding assays confirmed that chloroplast synthesized CTB binds to the intestinal membrane receptor of cholera toxin, indicating correct folding and disulfide bond formation within the chloroplast. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed. The introduced gene was stably inherited in the subsequent generation as confirmed by PCR and Southern blot analyses. Incrased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts

机译：转基因叶绿体技术可以提供生产胰岛素样生长因子I（IGF-I），人血清白蛋白（HSA）或干扰素（IFN）的可行解决方案，因为它具有超表达能力，折叠和加工真核蛋白的能力。二硫键（从而消除了对昂贵的纯化后处理的需求）。烟草是一种理想的选择，因为它具有大量的生物量，易于扩大规模（每棵植物上百万个种子），基因操纵以及迫切需要探索这种有害作物的替代用途。因此，所有三种人类蛋白质将表达如下：a）开发重组DNA载体以通过烟草叶绿体基因组增强表达'b）产生转基因植物c）使用分子和生化方法表征蛋白质或融合蛋白的转基因表达d）大规模从转基因烟草中纯化治疗性蛋白，并比较大肠杆菌或酵母中当前的纯化/加工方法e）用转基因烟草表征和比较酵母或大肠杆菌中产生的治疗性蛋白（产量，纯度，功能性）f）动物试验治疗性蛋白质有效性的临床前试验。可负担的疫苗的大量生产可以通过对植物进行基因工程以产生作为候选疫苗抗原的重组蛋白来实现。肠毒素性大肠杆菌（LTB）的B亚基和霍乱弧菌的霍乱毒素（CTB）是此类抗原的示例。当通过烟草核基因组表达天然LTB基因时，LTB积累的水平低于总可溶性叶蛋白的0.01％。在植物中产生有效水平的LTB，需要广泛的密码子修饰。叶绿体中未经修饰的CTB编码序列的扩增（每个细胞最多10,000个拷贝）导致高达总可溶性烟草叶蛋白寡聚体的积累达4.1％（表达水平比未经修饰的LTB基因高约410倍）。 PCR和Southern blot分析证实CTB基因稳定整合到叶绿体基因组中。 Western印迹分析表明，叶绿体合成的CTB组装成低聚物，并且与纯化的天然CTB在抗原上相同。同样，GM1-神经节苷脂结合测定法证实叶绿体合成的CTB与霍乱毒素的肠膜受体结合，表明叶绿体中正确的折叠和二硫键形成。与发育迟缓的核转基因植物相反，当组成型表达CTB时，叶绿体转基因植物在形态上与未转化的植物没有区别。通过PCR和Southern印迹分析证实，导入的基因在随后的世代中稳定遗传。在转基因叶绿体中增加了高效透粘膜载体分子和递送系统（如CTB）的生产

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公开/公告号IN239329B

专利类型
公开/公告日2010-03-19

原文格式PDF
申请/专利权人
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申请/专利号INPCT/2002/1089/KOL
发明设计人 DANIEL HENRY;
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申请日2002-08-23
分类号C12N15/21;C12N15/14;C12N15/82;C12N15/62;C12N1/00;C07K14/765;A01H5/00;C07K14/28;C12P21/02;C12N15/32;C12N15/16;C07K14/62;C12N15/17;C12N15/31;C12N15/20;
国家 IN
入库时间 2022-08-21 18:45:54

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