Related Experiment Videos
Identification and characterization of glycoprotein H of MDV-1 GA strain
Abstract:
A 2439 bp open reading frame (ORF) was identified from the DNA sequence of BamHI-F and -K2 fragments of Marek's disease virus of serotype 1 (MDV-1) GA strain, which predicts an 813 amino acid polypeptide. This peptide is homologous to HSV-1 gH, and has typical glycoprotein features. There are nine potential N-linked glycosylation sites within the extracellular domain. A fragment of the gH ORF was cloned into pGEX vector in frame with glutathione S-transferase (GST) to produce a GST-gH fusion protein in Escherichia coli. The GST-gH fusion protein was used to develop gH monoclonal and polyclonal antibodies. Expression of gH was detected in duck embryo fibroblasts (DEFs) infected with MDV-1 GA strain by immunofluorescence assay (IFA) with these antibodies. Virus neutralization and plaque-forming inhibition analyses were conducted with the gH antiserum. There were no neutralization and plaque-forming inhibition activities of gH antiserum. Comparison of the DNA sequence of gH gene between GA and RB1B strains of MDV-1 revealed major difference in the upstream control elements of gH ORF.
Insights
Researchers identified the Marek
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Marek's disease virus (MDV) is a highly contagious oncogenic avian herpesvirus.
- The glycoprotein H (gH) is a conserved protein in herpesviruses, often involved in viral entry and cell-to-cell spread.
- Understanding MDV gH function is crucial for developing effective control strategies against Marek's disease.
Purpose of the Study:
- To characterize the Marek's disease virus serotype 1 (MDV-1) glycoprotein H (gH) gene and its protein product.
- To generate antibodies against MDV-1 gH for expression analysis.
- To investigate the role of gH in viral neutralization and plaque formation.
Main Methods:
- Sequence analysis of the MDV-1 GA strain to identify the gH open reading frame (ORF).
- Cloning of a gH fragment for expression as a glutathione S-transferase (GST) fusion protein in Escherichia coli.
- Production of monoclonal and polyclonal antibodies against the GST-gH fusion protein.
- Immunofluorescence assay (IFA) to detect gH expression in infected duck embryo fibroblasts (DEFs).
- Virus neutralization and plaque-forming inhibition assays using gH antiserum.
Main Results:
- A 2439 bp ORF encoding an 813 amino acid polypeptide homologous to HSV-1 gH was identified.
- The gH protein possesses typical glycoprotein features, including nine potential N-linked glycosylation sites.
- MDV-1 gH expression was confirmed in infected DEFs using IFA.
- The generated gH antiserum showed no activity in virus neutralization or plaque-forming inhibition assays.
- Significant differences in upstream control elements of the gH gene were observed between MDV-1 GA and RB1B strains.
Conclusions:
- The MDV-1 gH gene has been identified and characterized, with its protein product expressed in infected cells.
- The lack of neutralizing activity suggests that gH may not be a primary target for humoral immunity in MDV-1 infection, or that other factors are involved.
- Sequence variations in regulatory regions may influence gH expression levels between different MDV-1 strains.