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Plant molecular farming for the production of industrial enzyme and vaccines.

机译:植物分子农业,用于生产工业酶和疫苗。

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摘要

Since the realization of plant genetic modification, transgenic plants have been utilized as platform for the production of valuable recombinant proteins or also known as plant molecular farming (PMF). The plant offers an economical system to reduce cost, the possibility of large scale production and is free of pathogens. The plant also provides versatility o products to accumulate from industrial enzymes, peptides, and pharmaceuticals. Recent advancement of RNA as regulator of gene expression and antiviral responses has shown a new area of research where transgenic plants are used to accumulate therapeutic RNAs. In this study, the reliability of transgenic plants as a platform for the production of industrial enzyme, subunit vaccine and therapeutic RNAs are being evaluated. Key factors that determine the success of molecular farming include 1) robustness of plant genetic transformation, 2) accumulation of large levels of recombinant product, 3) versatility of products to be accumulated, 4) stability of products, and 5) bioactivity of plant-derived products.;The first study demonstrated the accumulation of starch-degrading enzyme, amylopullulanase (APU) derived from bacteria Thermoanaerobacter thermohydrosulfuricus in maize seed. Accumulation of thermostable APU in transgenic maize seed resulted in direct starch processing into glucose and simple sugars without the addition of exogenous enzyme. Subsequent fermentation reaction showed 40.2% conversion of starch into bioethanol. This study showed simplification of starch-based bioethanol production from maize grains using a biotechnology approach.;The feasibility of the plant as the production system for the accumulation of antigen is demonstrated in the second study. Conserved nucleoprotein (NP) gene was expressed in maize seed. This conserved protein showed potential application as a universal vaccine for influenza virus. Our study suggested that maize-derived NP was immunogenic and could induce antibody responses upon administration in pigs and mice. Maize NP seeds remained stable and active at room temperature. This result showed that the plant is able to produce product and maintain immunogenicity.;Thirdly, the plant was utilized as a production system for the accumulation of dsRNA as a vaccine for shrimp viral pathogen. Our result showed the accumulation of long dsRNAs specific to white spot syndrome virus (WSSV) and infectious myonecrosis virus (IMNV). Shrimp bioassay of soybean-derived dsRNA specific to WSSV showed moderate protection against viral challenge and indicated activity of plant-derived dsRNA. While the accumulation levels of dsRNAs in transgenic plants was far from economically efficient, our result showed that the plant was able to accumulate foreign dsRNA. Our result showed that accumulation level needs to be improved for plant use as an efficient platform for accumulation of therapeutics RNA.;Concern over antibiotic or herbicide resistance selectable marker has brought attention for an alternative selectable marker. In the last study, a non-antibiotic selectable marker was assessed as a selection system for maize Agrobacterium-mediated transformation. The ptxD gene conferring phosphite oxidoreducatase enzyme enables transformed cells to utilize phosphite as phosphorous source while non-transformed cells cannot. Phosphite selection resulted in positive identification of transgenic events. The present study shows the feasibility and reliability of plants as a production system for a variety of products from enzyme, antigen and dsRNA.
机译:自实现植物遗传修饰以来,转基因植物已被用作生产有价值的重组蛋白的平台,或者也被称为植物分子耕作(PMF)。该工厂提供了一种经济的系统,可降低成本,大规模生产的可能性,并且不含病原体。该工厂还提供多功能性产品,可从工业酶,肽和药物中积累。 RNA作为基因表达和抗病毒应答调节剂的最新进展显示出了一个新的研究领域,其中转基因植物被用于积累治疗性RNA。在这项研究中,正在评估转基因植物作为生产工业酶,亚基疫苗和治疗性RNA的平台的可靠性。决定分子耕种成功的关键因素包括:1)植物遗传转化的稳健性,2)大量重组产物的积累,3)待积累产物的多功能性,4)产物的稳定性以及5)植物的生物活性首次研究证明了玉米种子中嗜热厌氧细菌嗜热厌氧细菌淀粉降解酶淀粉淀粉酶(APU)的积累。转基因玉米种子中热稳定APU的积累导致淀粉直接加工成葡萄糖和单糖,而没有添加外源酶。随后的发酵反应显示淀粉转化为生物乙醇的40.2%。这项研究表明,使用生物技术方法可以简化玉米籽粒中淀粉基生物乙醇的生产。;第二项研究证明了该植物作为积累抗原的生产系统的可行性。保守的核蛋白(NP)基因在玉米种子中表达。这种保守的蛋白质显示出作为流感病毒通用疫苗的潜在应用。我们的研究表明,玉米来源的NP具有免疫原性,在猪和小鼠中施用后可诱导抗体反应。玉米NP种子在室温下保持稳定和活跃。该结果表明该植物能够产生产物并保持免疫原性。第三,该植物被用作dsRNA积累的生产系统,作为虾病毒病原体的疫苗。我们的结果表明,长的dsRNA积累了白斑综合症病毒(WSSV)和传染性心肌坏死病毒(IMNV)。 WSSV特异的大豆来源的dsRNA的虾生物测定显示出对病毒攻击的中等保护作用,并表明了植物来源的dsRNA的活性。尽管转基因植物中dsRNA的积累水平远非经济有效,但我们的结果表明该植物能够积累外来dsRNA。我们的结果表明,需要提高植物的积累水平,作为治疗性RNA积累的有效平台。;对抗生素或除草剂抗性选择标记的关注已引起人们对替代选择标记的关注。在最近的研究中,非抗生素选择标记被评估为玉米农杆菌介导的转化的选择系统。赋予亚磷酸氧化还原酶的ptxD基因使转化的细胞能够利用亚磷酸作为磷源,而未转化的细胞则不能。亚磷酸酯的选择导致转基因事件的积极鉴定。本研究显示了植物作为酶,抗原和dsRNA多种产品生产系统的可行性和可靠性。

著录项

  • 作者

    Nahampun, Hartinio.;

  • 作者单位

    Iowa State University.;

  • 授予单位 Iowa State University.;
  • 学科 Plant sciences.;Biology.
  • 学位 Ph.D.
  • 年度 2015
  • 页码 219 p.
  • 总页数 219
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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