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Cloning and in vitro expression of the measles virus matrix gene
Abstract:
A cDNA library was prepared from Vero cells infected with the Edmonston strain of measles virus. A number of viral specific cDNA clones were isolated and characterized by Northern blot hybridization analysis. A cDNA clone containing a 1500 base pair insert which hybridizes to a viral specific transcript of approximately 1500 nucleotides was subcloned into pSP64 and used as an in vitro transcription template. The resulting RNA transcript was translated in a cell-free system, giving rise to a polypeptide which comigrates on polyacrylamide gels with the authentic measles virus matrix protein and is immunoprecipitated with antisera specific for the matrix protein.
Insights
Researchers created a measles virus cDNA library to identify viral genes. They successfully isolated and characterized a clone encoding the measles virus matrix protein using in vitro transcription and translation.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Measles virus (MeV) is a significant human pathogen.
- Understanding MeV gene expression is crucial for developing antiviral strategies.
- The matrix protein (M) plays a key role in MeV assembly and budding.
Purpose of the Study:
- To isolate and characterize cDNA clones encoding MeV proteins.
- To identify the specific cDNA clone for the MeV matrix protein.
- To confirm the identity of the translated polypeptide as the MeV matrix protein.
Main Methods:
- Preparation of a complementary DNA (cDNA) library from measles virus-infected Vero cells.
- Isolation and characterization of viral-specific cDNA clones using Northern blot hybridization.
- Subcloning of a selected cDNA insert into a transcription vector (pSP64).
- In vitro transcription to generate RNA transcript.
- Cell-free translation of the RNA transcript.
- Analysis of the translated polypeptide by polyacrylamide gel electrophoresis and immunoprecipitation.
Main Results:
- A cDNA library yielded several viral-specific clones.
- One clone with a 1500 base pair insert hybridized to a ~1500 nucleotide viral transcript.
- In vitro transcription and translation produced a polypeptide.
- The synthesized polypeptide comigrated with authentic measles virus matrix protein on gels.
- The polypeptide was immunoprecipitated by measles virus matrix protein-specific antisera.
Conclusions:
- A cDNA clone encoding the measles virus matrix protein was successfully isolated and characterized.
- The in vitro expression system confirmed the identity of the matrix protein.
- This study provides a valuable tool for further investigation of measles virus matrix protein function and interactions.