Construction of an infectious pseudorabies virus recombinant expressing a glycoprotein gIII-β-galactosidase fusion protein |
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Authors: | Calvin L Keeler Jr Mary E Whealy and Lynn W Enquist |
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Institution: | Central Research and Development Department, E.I. du Pont de Nemours & Co., Experimental Station E328/B22, Wilmington, DE 19898, U.S.A. Tel. (302)772-3199 |
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Abstract: | An infectious herpesvirus mutant has been constructed in which a major structural envelope glycoprotein gene was replaced by a hybrid gene encoding a novel fusion protein consisting of the N-terminus of the viral glycoprotein joined to Escherichia coli β-galactosidase (ßGal). Specifically, we fused DNA encoding the first 157 amino acids of the structural glycoprotein gIII from pseudorabies virus strain Becker to the E. coli lacZ gene in a bacterial expression vector. The resulting hybrid gene was then used to replace the wild-type gIII gene in the virus by cotransfection of plasmid and viral DNA. The desired viral recombinants were identified by their inability to react with specific monoclonal antibodies that recognized only wild-type gIII protein. One such mutant virus, PRV-Z1, was chosen for further analysis. PRV-Z1 expressed a glycosylated gIII-ßGal fusion protein after infection of PK15 cells. The fusion protein has no demonstrable ßGal activity and, although glycosylated, remains sensitive to the enzyme endo-β-N-acetylglucosaminidase H, unlike the mature gIII gene product, indicating that the fusion protein was incompletely processed. |
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Keywords: | Herpesvirus recombinant DNA hybrid protein lacZ transfection protein localization glycosylation |
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