chloroplast transformation, CTB-NSP4 fusion protein, pentamer forms, GM1 Binding assay
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 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.
Master of Science (M.S.)
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology
Molecular Biology and Microbiology
Length of Campus-only Access
Masters Thesis (Open Access)
Kalluri, Anila, "Expression Of Cholera Toxin B Subunit-rotavirus Nsp4 Enterotoxin Fusion Protein In Transgenic Chloroplasts" (2005). Electronic Theses and Dissertations. 454.