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Published on: November 1, 2011
[Molecular cloning,nucleotides sequence and transient expression of S and M genome segment of hantavirus strain
1Institute of Virology, Chinese Academy of Preventive Medicine, Beijing 100052, China.
Background:
To sequence, analyze and express the nucleotide sequences of S and M segments of hantavirus strain 84Fli.
Methods:
S and M segments of hantavirus 84Fli strain were amplified by RT-PCR, the PCR products were cloned into plasmid pCR2.1-TOPOr. Three clones of each segment which have been sequenced were randomly selected. The coding region of S and M segments were amplified by PCR, and cloned into expressing vector pcDNA3.0. Transient expression of nucleocapsid protein, G1 and G2 glycoproteins in COS7 cells were performed by Lipofectin transfection. The expression of NP, G1 and G2 have been conformed by using immunofluorescence, Western blot and immuno-precipitation methods.
Results:
The full length S and M segments cDNA have been amplified and cloned. The S and M segments of hantavirus strain 84Fli contained 1 688 and 3 616 nucleotides respectively.
Conclusions:
Deduced amino acid sequences of NP and glycoproteins revealed high homologue to other Hantaan viruses. NP, G1 and G2 proteins of 84Fli can be transiently expressed in COS7 cells.
Insights
Nucleotide sequences of hantavirus strain 84Fli were analyzed, revealing high homology to other Hantaan viruses. Key proteins, including nucleocapsid (NP) and glycoproteins (G1, G2), were successfully expressed in COS7 cells.
Area of Science:
- Virology
- Molecular Biology
- Genomics
Context:
- Hantaviruses are a significant public health concern, causing severe respiratory and hemorrhagic diseases.
- Understanding the genetic makeup and protein expression of novel hantavirus strains is crucial for developing diagnostics and therapeutics.
Purpose:
- To sequence and analyze the S and M segments of the hantavirus 84Fli strain.
- To express the nucleocapsid (NP) and glycoproteins (G1, G2) of hantavirus 84Fli in a mammalian cell line.
Summary:
- The full-length cDNA of the S and M segments of hantavirus strain 84Fli were amplified, cloned, and sequenced, measuring 1,688 and 3,616 nucleotides, respectively.
- The deduced amino acid sequences showed high homology to other Hantaan viruses.
- Transient expression of NP, G1, and G2 proteins was successfully achieved in COS7 cells, confirmed by immunofluorescence, Western blot, and immunoprecipitation.
Impact:
- Provides foundational genetic data for hantavirus 84Fli.
- Demonstrates the feasibility of expressing key viral proteins for further immunological studies.
- Contributes to the broader understanding of hantavirus diversity and pathogenesis.
