Abstract:The objective of this present study was to construct eukaryotic expression vector for mouse β-defensin 1 (mBD1) and influenza A virus M2e fusion gene, as well as study the expression and immunity of fused protein mBD1-M2e in MDCK cells. The mBD1 and M2e genes were amplified from plasmids pcDNA3.1 (+)/mBD1 and pcDNA3.1 (+)/M2e respectively. And a polypeptide linker Gly4Ser was used to splice mBD1 and M2e genes by SOE-PCR to construct mBD1-M2e fusion gene. Then the mBD1-M2e fragment was inserted into eukaryotic expressing plasmid pcDNA3.1 (+) to construct pcDNA3.1 (+) /mBD1-M2e. Recombinant plasmid was identified by restriction enzymes digestion, PCR and sequencing analysis, then the pcDNA3.1 (+)/mBD1-M2e was transfected into MDCK cells by PolyFect Transfection Reagent. The expression of mBD1-M2e gene was detected by immunofluorescence assay, RT-PCR and lymphocytes multiplication test with MTT. Expression vector pcDNA3.1 (+)/mBD1-M2e was constructed successfully in this study. The fused protein could be detected in MDCK cells transfected with pcDNA3.1 (+)/mBD1-M2e and could stimulate lymphocyte proliferation. The construction of the eukaryotic expression vectors for mBD1-M2e fusion gene, pcDNA3.1 (+)/ mBD1-M2e, was completely successful and the mBD1-M2e fusion protein could be expressed in MDCK cells stably. This result established a solid foundation for further exploration in efficient and broad-spectrum DNA vaccine against influenza A virus.
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