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Measles virus haemagglutinin gene: cloning, complete nucleotide sequence analysis and expression in COS cells
Abstract:
A measles virus (Hallé strain) cDNA library was prepared by cloning virus-induced mRNA directly into the expression vector PCD. Clones corresponding to the measles virus haemagglutinin (HA) gene were isolated and one, PCD-HA-15, which corresponded to the complete mRNA sequence, was further characterized. After transfection into COS-7 cells, measles virus HA antigen was detected by immunofluorescence. The [35S]methionine-labelled HA protein from transfected cells was immunoprecipitated by both polyclonal and monoclonal measles virus antibodies. Analysis by SDS-polyacrylamide gel electrophoresis revealed that the PCD-HA-15 protein migrated in a manner identical to the virus-induced HA. Nucleotide sequence analysis established that the gene contained 1949 nucleotides [exclusive of poly(A)] and coded for a protein containing 617 amino acids. A single hydrophobic domain likely to represent the transmembrane region was identified at the N-terminus. A second overlapping reading frame coded for a protein containing 70 amino acids. This contained a short hydrophobic region (16 amino acids) and had two potential N-glycosylation sites. Comparison of the HA gene of the Hallé strain with the published sequence of the Edmonston strain showed that there was a high degree of conservation (99.3%).
Insights
Researchers cloned the measles virus haemagglutinin (HA) gene, confirming its function and high conservation between strains. This work advances understanding of measles virus genetics and potential vaccine development.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Measles virus is a significant human pathogen.
- Understanding the measles virus haemagglutinin (HA) gene is crucial for vaccine development and viral entry mechanisms.
Purpose of the Study:
- To clone and characterize the measles virus (Hallé strain) haemagglutinin (HA) gene.
- To analyze the functional expression and sequence of the HA protein.
- To compare the HA gene sequence with other measles virus strains.
Main Methods:
- Construction of a measles virus cDNA expression library using the PCD vector.
- Isolation and characterization of HA gene clones, including PCD-HA-15.
- Transfection of COS-7 cells and detection of HA antigen via immunofluorescence.
- Immunoprecipitation and SDS-polyacrylamide gel electrophoresis of [35S]methionine-labelled HA protein.
- Nucleotide sequence analysis of the HA gene.
Main Results:
- The complete measles virus HA mRNA sequence was cloned into PCD-HA-15.
- Measles virus HA antigen and protein were successfully expressed in transfected COS-7 cells.
- The expressed HA protein exhibited identical migration patterns to virus-induced HA.
- Sequence analysis revealed a 1949-nucleotide gene coding for a 617-amino acid protein with a transmembrane domain.
- A second overlapping reading frame coding for a 70-amino acid protein with N-glycosylation sites was identified.
- High sequence conservation (99.3%) was observed between the Hallé and Edmonston strains of the measles virus HA gene.
Conclusions:
- The cloned PCD-HA-15 construct effectively expresses functional measles virus HA antigen.
- The identified gene structure and protein characteristics provide insights into measles virus biology.
- The high conservation of the HA gene suggests its critical role and potential for cross-strain vaccine efficacy.