Structure and Expression of Genes for Flavivirus Immunogens.
Abstract
As the first step toward expression of full-length copies of the DEN- 1 E and PrM proteins in E. coli, cDNA copies of these proteins were amplified by PCR technology and cloned into T7 RNA polymerase expression vectors. Analysis revealed that while the PrM protein was successfully placed into the expression plasmid, and expressed under inducing conditions, an intact copy of the E protein could not be successfully cloned under any of a wide variety of protocols. Rather, plasmids containing all of the 5' half of the E coding sequence, and an undetermined portion of the 3' end of the sequence were repeatedly obtained. Our results indicated that even the truncated version of E could only be obtained in a low copy-number vector. The truncation event does not occur at the level of PCR amplification of the E protein sequence. The explanation for our failure to obtain full-length clones of the E protein is not readily apparent, as the expression vector employed was designed for expression of otherwise toxic genes, and no expression of cloned sequences should be taking place in the host strains used for plasmid constructions. Flaviviruses; Recombinant DNA; PCR Amplification; Subunit vaccine; Biotechnology, RA I, Dengue Virus.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 20, 1992
- Accession Number
- ADA252662
Entities
People
- Maurille J. Fournier
- Thomas L. Mason
Organizations
- University of Massachusetts Amherst