Hepatitis C viral infection is the major cause of acute hepatitis and chronic liver disease and remains the leading cause of liver transplants (NIH). An estimated 180 million people are infected globally (WHO). There is no vaccine available to prevent hepatitis C. The treatment with antiviral drugs is expensive, accompanied with various side effects and is limited only to those at risk of developing advanced liver disease. The treatment is also effective in only about 30% to 50% of treated patients and still a high percentage of patients are resistant to therapy. Therefore, there is an urgent need for the development of effective vaccine antigens and an efficacious HCV vaccine. The non-structural 3 protein of the hepatitis C virus is a multifunctional protein of the virus required for virus polyprotein processing and replication. Vaccine antigen production via chloroplast transformation system usually results in high expression levels and eliminates the possibility of contamination with vector sequences,human or animal pathogens. The HCV NS3 antigen was expressed in the chloroplast of Nicotiana tabacum var. Petit havana and LAMD-609. The 1.9kb NS3 gene was cloned into a chloroplast expression vector, pLD-Ct containing the 16S rRNA promoter, aadA gene coding for the spectinomycin selectable marker, psbA 5' untranslated region to enhance translation in the light and 3' untranslated region for transcript stability and trnI & trnA homologous flanking sequences for site specific integration into the chloroplast genome. Chloroplast integration of the NS3 gene was first confirmed by PCR. Southern blot analysis further confirmed site-specific gene integration and homoplasmy. The NS3 protein was detected in transgenic chloroplasts by immunoblot analysis. The NS3 protein was further quantified by ELISA. Maximum expression levels of NS3 up to 2% in the total soluble protein were observed even in old leaves, upon 3-day continuous illumination. These results demonstrate successful expression of the HCV non-structural 3 antigen in transgenic tobacco chloroplasts. Animal studies to test the immunogenecity of the chloroplast derived HCV NS3 will be performed using chloroplast derived NS3 antigen.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd-1283 |
| Date | 01 January 2005 |
| Creators | Bhati, Anubhuti |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations |
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