Temperature dependent characteristics of a recombinant infectious hematopoietic necrosis virus glycoprotein produced in insect cells

Diseases of Aquatic Organisms

Kenneth D. Cain¹  Alberta L. Brassfield¹  Scott E. LaPatra¹  Sandra S. Ristow¹  Katherine M. Byrne¹ 

关键词: IHNV;    G protein;    Baculovirus;    Recombinant;    Conformational alterations;    Monoclonal antibodies;   

DOI  :  10.3354/dao036001

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

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 wereevaluated 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 µg/1 x10⁵ 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 revealeda 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°C (RecG_low),immunostaining and cell fusion activity demonstrated surface localization of the protein. In contrast, when recombinant protein was produced at 27°C (RecG_high), G protein was sequestered within the cell, suggesting that at the 2 differenttemperatures 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 RecG_high andRecG_low under non-reducing conditions; however, 1 neutralizing MAb (92A) recognized RecG_low but failed to react to RecG_high under non-reducing conditions. This suggests that differences exist between RecG_low andRecG_high which may have implications in the development of a properly folded recombinant G protein with the ability to elicit protective immunity in fish.

【 授权许可】

Unknown