Expression of Dengue 3 Envelope and Non-Structural 1 Glycoproteins in the Fungus Neurospora Crassa. Phase 1.
Abstract
The four serotypes of dengue viruses are a leading cause of morbidity throughout the tropics and subtropics. For use in subunit vaccines and for diagnostics, a reliable and inexpensive source of dengue proteins is needed. We have evaluated the ability of the filamentous fungus Neurospora crassa to produce authentic dengue 3 envelope (E) and nonstructural 1 (NS1) glycoproteins. We synthesized E and NS1 cDNA fragments from total RNA extracted from DEN-3 infected mosquito cells using oligonucleotide primers and a reverse transcription-PCR amplification method. The use of PCR technology allowed us to precisely define the ends of the gene fragments and provided a means to introduce unique restriction sites, start and stop translational codons, and the Neurospora translational consensus sequence. Neurospora expression plasmids were constructed by subcloning the dengue gene fragments between a constitutive promoter and a transcriptional termination sequence. Following addition of a dominant, selectable marker to the expression plasmids, we transformed Neurospora and verified integration of the E and NS1 sequences in transformers by Southern blot analysis. We were unable, however, to detect dengue proteins expressed from this constitutive promoter on Western blots of either secreted or intracellular proteins prepared from transformants.
Document Details
- Document Type
- Technical Report
- Publication Date
- Oct 19, 1990
- Accession Number
- ADB160775
Entities
People
- John M. Ivy