Molecular cloning and analysis of the infectious hematopoietic necrosis virus genome and development of a subunit vaccine Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/np193c54g

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  • Complementary DNA (cDNA) clones were generated from the RNA genome of infectious hematopoietic necrosis virus (IHNV) by using random DNA oligomers to prime first strand synthesis. These clones were mapped to their respective locations on the genome and used to determine the nucleotide sequence of the nucleocapsid (N) gene and the intergenic region between the glycoprotein (G) and the nonvirion (NV) genes. Interesting features of the N gene sequence include short homologies with N genes of other rhabdoviruses at the 5' and 3' non-coding termini of the mRNA, as well as an exceptionally long 5' non-translated region of the mRNA suggesting a leader RNA may be coupled to the N mRNA. The IHNV N protein coding region shows no significant homology with other rhabdovirus N genes at either the nucleotide or amino acid level. The intergenic region between the G and NV genes also shares some sequence homology with those reported for other negative strand RNA viruses. In addition, plasmid vectors were constructed which expressed an antigenic determinant of the glycoprotein gene of IHNV as a fusion protein with the trpE protein of Escherichia coli. Insertion of Sau3AI fragments from the IHNV glycoprotein gene into trpE expression plasmids led to a fusion protein containing a hydrophilic segment of 104 amino acids from the middle portion of the viral glycoprotein. After induction with indoleacrylic acid, fusion proteins accumulated stably in the E. coli cells and accounted for approximately 10% of the total protein in the cell. Immunization trials in fish with the crude bacterial lysate containing the fusion protein have indicated that the trpE-glycoprotein fusion protein produced in bacteria does induce protective immunity.
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