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Characterization of baculovirus p10 synthesis using monoclonal antibodies
Abstract:
A series of monoclonal antibodies were produced against virion proteins of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV). Four of these antibodies reacted with a protein of 14 kd on Western blots of electrophoretically separated OpMNPV virion proteins. These antibodies were used to identify immunoreactive clones from a lambda gt11 expression library of OpMNPV DNA. By hybridization of insert DNA from the lambda gt11 clones to blots of digests of OpMNPV genomic DNA, and by sequencing the ends of the lambda gt11 inserts, these clones were shown to contain a portion of the p10 gene. The regions containing epitopes recognized by the four monoclonal antibodies were located using fusion proteins made from selected portions of the p10 reading frame in a trpE vector. One of the p10 antibodies was used to characterize p10 synthesis in infected Lymantria dispar cells by using Western blots and immunofluorescent staining. The p10 protein was detected with immunofluorescent microscopy at 14 hr postinfection and by 20 hr it formed intensely staining cytoplasmic structures. On Western blots of infected cells, two forms of p10 (of about 14 and 15 kd) were observed. One of the p10 monoclonal antibodies showed a strong cross-reaction with cytoskeletal structures in uninfected insect cells and rat fibroblasts.
Insights
Monoclonal antibodies were developed against Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) virion proteins. These antibodies helped identify clones containing part of the p10 gene and characterized p10 protein synthesis in infected cells.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Monoclonal antibodies (mAbs) are crucial tools for identifying specific viral proteins.
- Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) is an important insect pathogen.
Purpose of the Study:
- To produce and characterize monoclonal antibodies against OpMNPV virion proteins.
- To identify and analyze the p10 gene and its protein product.
- To investigate the synthesis and localization of the p10 protein during viral infection.
Main Methods:
- Production and screening of monoclonal antibodies against OpMNPV.
- Western blotting and immunofluorescent staining for protein detection.
- Lambda gt11 expression library screening and DNA sequencing.
- Analysis of p10 protein expression in infected insect cells.
Main Results:
- Four mAbs recognized a 14 kDa OpMNPV virion protein.
- These mAbs identified lambda gt11 clones containing a portion of the p10 gene.
- p10 protein synthesis was detected in infected Lymantria dispar cells starting at 14 hr post-infection.
- Two forms of p10 (14 and 15 kDa) were observed in infected cells.
- One mAb cross-reacted with cytoskeletal structures in uninfected cells.
Conclusions:
- The developed monoclonal antibodies are specific tools for studying OpMNPV.
- The p10 gene and its protein product were characterized.
- p10 protein expression and localization dynamics were elucidated during OpMNPV infection.
- Potential cross-reactivity of p10 antibodies with host cell structures was noted.