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- Title
- RECEPTOR MEDIATED ORAL DELIVERY OF BIOENCAPSULATED GREEN FLUORESCENT PROTEIN EXPRESSED IN TRANSGENIC CHLOROPLASTS.
- Creator
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Limaye, Arati, Daniell, Henry, University of Central Florida
- Abstract / Description
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The skyrocketing costs of prescription medicine in developed countries and their lack of availability in developing countries are the most challenging problems of human health. Primary reasons for such high cost are fermentation-based production, expensive purification methods, the need for low temperature storage and transportation and the delivery through sterile injections. Most of these expenses could be minimized or eliminated when therapeutic proteins are expressed and orally delivered...
Show moreThe skyrocketing costs of prescription medicine in developed countries and their lack of availability in developing countries are the most challenging problems of human health. Primary reasons for such high cost are fermentation-based production, expensive purification methods, the need for low temperature storage and transportation and the delivery through sterile injections. Most of these expenses could be minimized or eliminated when therapeutic proteins are expressed and orally delivered via plant cells. Chloroplasts have the machinery to fold complex and biologically active eukaryotic proteins in the soluble chloroplast stromal compartment. Protein expression through chloroplast transformation system offers a number of advantages over nuclear transformation such as a high level of transgene expression (up to 47% of the total soluble protein), due to the presence of 10,000 copies of the transgene per cell, which is uniquely advantageous for oral delivery of adequate amounts of the therapeutic protein or vaccine antigen. It is also an environmentally friendly approach due to effective gene containment and lack of transgene expression in pollen since the chloroplast genome is maternally inherited. To study receptor-mediated oral delivery of therapeutic proteins using the transmucosal carrier cholera toxin B subunit (CTB), a CTB-GFP fusion protein separated by a furin cleavage site was expressed via the tobacco chloroplast genome and used as a visible marker. Site specific integration of the transgene was confirmed by PCR analysis. Southern blot analysis confirmed homoplasmy. Immunoblot analysis confirmed the expression of both the monomeric as well as the pentameric forms of CTB-GFP in transgenic plants. Expression levels of upto 21.3% were obtained and the functionality of the CTB-GFP pentamers was confirmed by an in vitro GM1 binding assay. GFP was seen in the intestinal mucosa, liver and spleen of mice orally fed with CTB-GFP expressing leaves, while CTB was detected only in the intestinal cells. Intestinal macrophages and dendritic cells stained positive for both the CTB as well as GFP. These results suggest successful cleavage of the foreign protein from the transmucosal carrier and its delivery to various organs. These investigations should facilitate the development of a novel cost-effective oral delivery system for plant-derived therapeutic proteins.
Show less - Date Issued
- 2005
- Identifier
- CFE0000891, ucf:46633
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000891
- Title
- EXPRESSION OF CHOLERA TOXIN B SUBUNIT-ROTAVIRUS NSP4 ENTEROTOXIN FUSION PROTEIN IN TRANSGENIC CHLOROPLASTS.
- Creator
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Kalluri, Anila, Daniell, Henry, University of Central Florida
- Abstract / Description
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Rotavirus, the major cause of life-threatening infantile gastroenteritis, is a member of the Reoviridae family and is considered to be the single most important cause of virus-based severe diarrheal illness in infants and young children particularly 6 months to 2 years of age in industrialized and developing countries. Infection in infants and young children is often accompanied by severe life threatening diarrhea, most commonly following primary infection. Diarrhea is the major cause of...
Show moreRotavirus, the major cause of life-threatening infantile gastroenteritis, is a member of the Reoviridae family and is considered to be the single most important cause of virus-based severe diarrheal illness in infants and young children particularly 6 months to 2 years of age in industrialized and developing countries. Infection in infants and young children is often accompanied by severe life threatening diarrhea, most commonly following primary infection. Diarrhea is the major cause of death among children around the world. Responsible for 4 to 6 million deaths per year according to the World Health Organization (WHO), diarrhea is especially dangerous for infants and young children. Globally, it is estimated that 1.4 billion episodes of diarrhea occur in children less than five years of age annually. In the United States alone, rotavirus causes more than 3 million cases of childhood diarrhea each year, leading to an estimated 55,000 to 100,000 hospitalizations and 20 to 100 deaths. And is a major cause of mortality for children in developing countries with approximately one million deaths annually. Rotaviruses belong to the family Reoviridae and are spherical 70-nm particles. The virus genome contains 11 segments of double-stranded RNA, each encoding a viral capsid or nonstructural protein. The identification of a rotavirus nonstructural protein gene (NSP4) encoding a peptide, which functions both as a viral enterotoxin and as a factor involved in the acquisition of host cell membrane during virus budding from cells, provides a new approach for mucosal immunization. Protein expression through chloroplast transformation system offers a number of advantages like high level of transgene expression, transgene containment via maternal inheritance, lack of gene silencing and position effect due to site specific gene integration and also the possibility of multi gene engineering in single transformation event. It is also an environmentally friendly approach due to effective gene containment and lack of transgene expression in pollen. To achieve an enhanced immune response to rotavirus infection, a fusion gene encoding the cholera toxin B subunit linked to rotavirus enterotoxin 90 aa protein (CTB-NSP490) was introduced into transgenic chloroplast and was transformed into chloroplast genome of Nicotiana tabacum by homologous recombination. The chloroplast integration of CTB-NSP4(90) fusion gene was confirmed in transgenic tobacco plants by PCR analysis. Southern blot analysis further confirmed site specific gene integration and homoplasmy. Immunoblot analysis of transformed chloroplast confirmed the expression of CTBNSP490 fusion protein both in monomeric and pentameric forms that retained the binding affinity to the enterocytes GM1 ganglioside receptor. Expression levels of CTB-NSP4 protein was quantified by GM1 ganglioside binding ELISA assay; mature leaves expressed CTB-NSP4 fusion protein to upto 2.45 % in total soluble protein, 100-400 fold higher than nuclear expression which was only 0.006%-0.026%. Antibody titration and virus challenge experiments will be performed in mice at Loma Linda University to evaluate the antigenic and protective properties of the chloroplast derived CTB-NSP4 fusion protein.
Show less - Date Issued
- 2005
- Identifier
- CFE0000655, ucf:46540
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000655
