title: Temperature dependent characteristics of a recombinant infectious hematopoietic necrosis virus glycoprotein produced in insect cells.
abstract: A recombinant infectious hematopoietic necrosis virus (IHNV) glycoprotein (G protein) was produced in insect cells using a baculovirus vector (Autographa californica nuclear polyhedrosis virus). Characteristics of this protein were evaluated in relation to native viral G protein. A full-length (1.6 kb) cDNA copy of the glycoprotein gene of IHNV was inserted into the baculovirus vector under control of the polyhedrin promoter. High levels of G protein (approximately 0.5 microgram/1 x 10(5) cells) were produced in Spodoptera frugiperda (Sf9) cells following recombinant baculovirus infection. Analysis of cell lysates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot revealed a recombinant IHNV G of slightly higher mobility on the gel than the viral G protein. Differences in mobility were abrogated by endoglycosidase treatment. When the recombinant G protein was produced in insect cells at 20 degrees C (RecGlow), immunostaining and cell fusion activity demonstrated surface localization of the protein. In contrast, when recombinant protein was produced at 27 degrees C (RecGhigh), G protein was sequestered within the cell, suggesting that at the 2 different temperatures processing differences may exist. Eleven monoclonal antibodies (MAbs) were tested by immunoblotting for reactivity to the recombinant G protein. All 11 MAbs reacted to the reduced proteins. Four MAbs recognized both RecGhigh and RecGlow under non-reducing conditions; however, 1 neutralizing MAb (92A) recognized RecGlow but failed to react to RecGhigh under non-reducing conditions. This suggests that differences exist between RecGlow and RecGhigh which may have implications in the development of a properly folded recombinant G protein with the ability to elicit protective immunity in fish.
authors.affiliation: Department of Animal Sciences, Washington State University, Pullman 99164, USA.
authors.forename: K D
authors.initials: KD
authors.lastname: Cain
authors.forename: K M
authors.initials: KM
authors.lastname: Byrne
authors.forename: A L
authors.initials: AL
authors.lastname: Brassfield
authors.forename: S E
authors.initials: SE
authors.lastname: LaPatra
authors.forename: S S
authors.initials: SS
authors.lastname: Ristow
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000737:chemistry; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005393:Fish Diseases / Q000276:immunology / Q000517:prevention & control / Q000821:virology; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D005966:Glucuronidase / Q000737:chemistry; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006026:Glycoside Hydrolases / Q000737:chemistry; D006358:Hot Temperature; D008856:Microscopy, Fluorescence; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000378:metabolism; D017686:Oncorhynchus mykiss; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012209:Rhabdoviridae / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018353:Rhabdoviridae Infections / Q000276:immunology / Q000517:prevention & control / Q000662:veterinary; D012474:Salmon; D018411:Spodoptera; D014614:Vaccines, Synthetic / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D014760:Viral Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014765:Viral Vaccines / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D006023:Glycoproteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D014760:Viral Fusion Proteins; D014765:Viral Vaccines; D006026:Glycoside Hydrolases; D005966:Glucuronidase