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首页> 外文学位 >The production of cervical cancer-related HPV16 E7 oncoprotein in rice seeds.
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The production of cervical cancer-related HPV16 E7 oncoprotein in rice seeds.

机译：水稻种子中与宫颈癌相关的HPV16 E7癌蛋白的产生。

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摘要

Cervical cancer is a leading cause of cancer-related death in women of developing countries and the second most prevalent cancer among women of developed countries. HR-HPV (high-risk human papillomavirus) type 16 is mainly considered as the causative agent of more than half of clinical cervical cancer cases, and the E7 oncoprotein of HPV16 is responsible for causing and maintaining the transformed state of tumor cells. HPV16 E7 is regarded as a tumor-specific antigen because of continual expression in cancer cells, and hence is a promising target for a therapeutic vaccine against cervical cancer. Since the last decade, plant-based expression systems have been promoted for large-scale production of recombinant proteins of pharmaceutical, industrial and agricultural applications. In this study, HPV16 E7 is produced in rice seeds by manipulating the secretory pathway and targeting endomembrane system organelles of rice endosperm cells for better yield and stability. Large numbers of independent transformants of secretory expression cassettes (pE7 and p←E7) and vacuolar expression cassette (pE7CTPP), expressing recombinant E7 under the control of the rice glutelin promoter and its signal peptide were produced by Agrobacterium transformation of Oryza sativa L. cv. Nipponbare. The expression of recombinant E7 was confirmed by Western blot analysis and ELISA (enzyme linked immunosorbent assay). The pE7 and p←E7 transformants, differing with respect to the proximity of glutelin promoter to CaMV 35S promoter (governing expression of selectable marker gene), accumulate recombinant E7 to similar levels. Transformants of pE7CTPP, containing the rice lectin CTPP (carboxyl terminal propeptide) for vacuolar targeting of recombinant E7, accumulate recombinant E7 to a level 3-4 fold more than that in pE7 and p←E7 transformants. Non-cleavage of CTPP from recombinant E7 (E7-CTPP) was observed, and suggests the inappropriateness of utilization of a CTPP for vacuolar targeting of recombinant proteins of pharmaceutical importance. The recombinant E7 was present predominantly in an insoluble form of high-oligomeric order in rice endosperm cells. In the pE7 and p←E7 transformants, the recombinant E7 was engineered to follow the default pathway to the intercellular space of rice endosperm cells. However, fluorescence and electron microscopy showed that recombinant E7 was deposited in PSVs (protein storage vacuoles) and prolamin protein bodies. When rice lectin CTPP was utilized in addition to the glutelin signal peptide, the recombinant E7-CTPP exhibited a similar localization pattern to PSVs (protein storage vacuoles) and prolamin protein bodies. Thus, the specialized nature of storage tissue appears to influence the recombinant protein trafficking and localization.
机译：宫颈癌是发展中国家妇女与癌症有关的死亡的主要原因，也是发达国家妇女中第二大流行的癌症。 HR-HPV（高危型人乳头瘤病毒）16型主要被认为是一半以上临床子宫颈癌病例的病原体，HPV16的E7癌蛋白负责引起并维持肿瘤细胞的转化状态。 HPV16 E7由于在癌细胞中持续表达而被认为是肿瘤特异性抗原，因此是针对宫颈癌的治疗性疫苗的有希望的靶标。自上个十年以来，基于植物的表达系统已得到推广，可用于制药，工业和农业应用的重组蛋白的大规模生产。在这项研究中，通过操纵分泌途径并靶向水稻胚乳细胞的内膜系统细胞器，可以在水稻种子中产生HPV16 E7，从而获得更好的产量和稳定性。在水稻谷蛋白启动子的控制下，表达大量重组E7的分泌表达盒（pE7和p←E7）和液泡表达盒（pE7CTPP）的大量独立转化体是通过农杆菌转化稻米产生的。 。 Nipponbare。通过Western印迹分析和ELISA（酶联免疫吸附测定）确认重组E7的表达。 pE7和p←E7转化子在谷蛋白启动子与CaMV 35S启动子的接近性（选择性标记基因的表达）方面有所不同，将重组E7积累到相似的水平。含有水稻凝集素CTPP（羧基末端前肽）的液泡靶向重组E7的pE7CTPP转化子积累的重组E7含量比pE7和p←E7转化子高3-4倍。观察到未从重组E7（E7-CTPP）裂解CTPP，这表明利用CTPP进行液泡靶向具有重要医药意义的重组蛋白是不合适的。重组E7主要以不溶形式存在于水稻胚乳细胞中的高寡聚体。在pE7和p←E7转化子中，重组E7被设计为遵循默认途径进入水稻胚乳细胞的细胞间空间。但是，荧光和电子显微镜显示重组E7沉积在PSV（蛋白储存液泡）和醇溶蛋白蛋白体内。当使用谷蛋白凝集素信号肽以外的水稻凝集素CTPP时，重组E7-CTPP表现出与PSV（蛋白储存液泡）和谷醇溶蛋白蛋白体相似的定位模式。因此，存储组织的特殊性质似乎影响重组蛋白的运输和定位。

著录项

  • 作者

    Mehra, Amit.;

  • 作者单位

    University of Arkansas.;

  • 授予单位 University of Arkansas.;
  • 学科 Biology Molecular.;Agriculture Plant Culture.;Biology Cell.
  • 学位 Ph.D.
  • 年度 2008
  • 页码 146 p.
  • 总页数 146
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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