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Chloroplast-Derived Vaccine Antigens and Biopharmaceuticals: Protocols for Expression, Purification, or Oral Delivery and Functional Evaluation

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Recombinant Proteins From Plants

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 483))

Summary

Many vaccine antigens and biopharmaceutical proteins have been expressed at high levels via the chloroplast genome and their functionality has been evaluated using in vitro assays in cell cultures (i.e., macrophage lysis assay, inhibition of vesicular stomatitis virus-induced cytopathicity in baby hamster kidney cells, or inhibition of human HIV infection in TZM-BL cells) as well as protection after challenge with bacterial or viral pathogens or antitumor assays or delay the onset of insulitis in suitable animal models. Production of therapeutic proteins in chloroplasts eliminates the expensive fermentation technology. Moreover, oral delivery of chloroplast-derived therapeutic proteins eliminates expensive purification steps, cold storage, cold transportation, and delivery via sterile needles, thereby further decreasing their cost. In this chapter, we describe detailed protocols for chloroplast transformation including the construction of chloroplast transformation vectors, delivery of DNA into plant cells using particle bombardment, selection and regeneration of transformants by tissue culture, confirmation of transgene integration into the chloroplast genome and homoplasmy, evaluation of foreign gene expression, purification of foreign protein, or oral delivery via bioencapsulation, functional evaluation using in vitro and in vivo assays, and evaluation of immunity after challenge with pathogens in suitable animal models.

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Acknowledgment

The protocols reported in this article were supported in part by grants from USDA 3611-21000-021-02S and NIH R01 GM 63879 to Henry Daniell.

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Authors and Affiliations

  1. N. Department of Molecular Biology and Microbiology, University of Central Florida, Biomolecular Science, Orlando, FL, U.S.A.

    N. Dolendro Singh

  2. Department of Molecular Biology and Microbiology, University of Central Florida, Biomolecular Science, FL, U.S.A.

    Yi Ding

  3. Department of Molecular Biology and Microbiology, University of Central Florida, Biomolecular Science, Orlando, FL, U.S.A.

    Henry Daniell

Authors
  1. N. Dolendro Singh
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  2. Yi Ding
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  3. Henry Daniell
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Editors and Affiliations

  1. CNRS, Université de Rouen, Mont Saint Aignan, France

    Loïc Faye  & Véronique Gomord  & 

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Singh, N.D., Ding, Y., Daniell, H. (2009). Chloroplast-Derived Vaccine Antigens and Biopharmaceuticals: Protocols for Expression, Purification, or Oral Delivery and Functional Evaluation. In: Faye, L., Gomord, V. (eds) Recombinant Proteins From Plants. Methods in Molecular Biology™, vol 483. Humana Press. https://doi.org/10.1007/978-1-59745-407-0_10

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  • DOI: https://doi.org/10.1007/978-1-59745-407-0_10

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-978-9

  • Online ISBN: 978-1-59745-407-0

  • eBook Packages: Springer Protocols

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