Cloning and Expression of Genes for Dengue Virus Type-2 Encoded-Antigens for Rapid Diagnosis and Vaccine Development
Abstract
We carried out DNA sequence analysis of dengue virus type 2 cDNA clones 505, A4, and B2, which were cloned in our laboratory at KUMC. In addition, we sequenced the cDNA clone, pRP2 which was cloned by Dr. Robert Putnak at WRAIR. The pRP2 clone encodes the region from the carboxy terminal region of E glycoprotein to the N- terminal region of NS2A. The sequence data obtained by Maxam-Gilbert method in our laboratory was compared with the data obtained by Sanger's dideoxy sequencing method. A manuscript resulting from this collaborative work has been submitted to Virology. The complete sequence of clones 505, A4, and B2 gave rise to a total of 7406 nucleotides, so far sequenced of dengue virus type 2 genome (New Guinea strain). We chemically synthesized specific oligodeoxynucleotide primers, based on our sequence data. These primers are currently being used to complete our cDNA cloning and DNA sequence analysis. We have expressed the cDNA encoding different antigens of dengue-2 virus in E. coli as fusion protein to beta-galactosidase. The purified protein was used to immunize rabbits to produce polyclonal antisera. The recombinant fusion polypeptides were analyzed by SDS-polyacrylamide for their sizes and immunoreactivities with the polyclonal mouse ascites hyper immune sera, as well as, with the monoclonal antisera against NS1 and E. The fusion constructs expressing E and NS1 were verified by DNA sequence analysis to map the region which is fused with the reading frame encoding beta-galactosidase.
Document Details
- Document Type
- Technical Report
- Publication Date
- Nov 17, 1987
- Accession Number
- ADA203472
Entities
People
- Radha K. Padmanabhan
Organizations
- University of Kansas Medical Center