Graduate Thesis Or Dissertation
 

Cloning, sequence and expression of the glycoprotein gene of infectious hematopoietic necrosis virus, a fish rhabdovirus

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  • Double-stranded cDNA was prepared from infectious hematopoietic necrosis virus mRNA and cloned into the plasmid vector pUC8. A cDNA clone (pG8) containing the entire coding region for the glycoprotein (G-protein) of infectious hematopoietic necrosis virus was selected by hybridization to a ³²P-labeled probe. The restriction map and nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined using this full-length cDNA clone. The G-protein cDNA is 1,609 nucleotides long (excluding the polyadenylic acid) and encodes a protein of 508 amino acids. The predicted amino acid sequence was compared with that of the glycoprotein of the Indiana and New Jersey serotypes of vesicular stomatitis virus and with the glycoprotein of rabies virus, using a computer program which determined optimal alignment. An amino acid identity of approximately 20% was found between infectious hematopoietic necrosis virus and the two vesicular stomatitis virus serotypes and between infectious hematopoietic necrosis virus and rabies virus. The positions and sizes of the signal sequence and transmembrane domain and the possible glycosylation sites were determined. A cDNA fragment containing the gene encoding the glycoprotein of infectious hematopoietic necrosis virus was inserted into Autographa califomica Baculovirus vectors under the control of the polyhedrin promoter. Insect cells were cotransfected with the Baculovirus recombinants and wild-type Autographa califomica nuclear polyhedrosis virus genomic DNA. Recombinant viruses were selected by plaque assay and plaque hybridization. Gel electrophoresis and Western blot analysis revealed the presence of a 66 kilodalton protein identical in size to the glycoprotein of infectious hematopoietic necrosis virus.
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  • File scanned at 300 ppi (Monochrome, 8-bit Grayscale) using ScandAll PRO 1.8.1 on a Fi-6670 in PDF format. CVista PdfCompressor 4.0 was used for pdf compression and textual OCR.
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