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[Establishment of mammalian cell lines for constitutive expression of influenza virus matrix protein 2]
Ai-Jun Chen1, Jian-Qiang Guo, Li-Hong Yao
1Molecular Virology and Genetic Engineering Laboratory, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China. chenajsun@yahoo.com.cn
Abstract:
To establish a mammalian cell line for stable expression of the matrix protein 2 (M2) of influenza virus type A. M2 gene was amplified by PCR from the influenza virus strain A/PR/8/34. The PCR product was cloned into eukaryotic expression vector pcDNA5/FRT. After identification with restriction enzyme digestion, the plasmid was co-transfected with plasmid pOG44 which expressed Flp in Flp-In-CHO cells. The target gene was integrated into chromosome of CHO cells by homologous recombination in vivo. Recombinant CHO-M2 cell lines were selected for hygromycin B resistance. A total of 15 recombinant cell strains with high expression of M2 protein were screened by hygromycin, and the expression of M2 protein was determined by IFA and Western blot. After subculturing for 10 passages, the presence of M2 gene in the CHO-M2 cells was confirmed by PCR, and the expression of M2 protein were proved by IFA and Western blot. We successfully constructed a mammalian cell line which stably expressed M2 protein of influenza virus type A. The cell line will be useful for studies on function of M2 protein and provide tools for novel influenza virus vaccine development.
Insights
Researchers created a stable mammalian cell line for expressing the influenza A virus matrix protein 2 (M2). This new cell line aids in studying M2 protein function and developing new influenza vaccines.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Context:
- Influenza A virus matrix protein 2 (M2) is crucial for viral assembly and ion channel function.
- Developing stable mammalian cell lines for viral protein expression is essential for research and therapeutic development.
Purpose:
- To establish a Flp-In-CHO mammalian cell line for stable expression of the influenza A virus M2 protein.
- To characterize the M2-expressing cell line for its stability and protein expression levels.
Summary:
- The M2 gene from influenza A virus strain A/PR/8/34 was amplified and cloned into the pcDNA5/FRT vector.
- Co-transfection with pOG44 in Flp-In-CHO cells facilitated homologous recombination and stable integration of the M2 gene into the CHO cell chromosome.
- Recombinant cell lines were selected using hygromycin B resistance, and M2 protein expression was confirmed via immunofluorescence assay (IFA) and Western blot, demonstrating stable expression over 10 passages.
Impact:
- Successfully constructed a stable mammalian cell line expressing influenza A virus M2 protein.
- This cell line serves as a valuable tool for investigating M2 protein functions.
- Provides a platform for the development of novel influenza virus vaccines.
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