Construction of a recombinant bacmid DNA in order to express Neuraminidase gene of influenza virus H1N1

In recent years Influenza viruses have caused widely spread moderate to severe infection in all around the world and there is no Influenza vaccine which can protect people only with one dose injection till now. Therefore، producing a universal vaccine based on virus like particle (VLP) could be ideal. In this study one of the molecular structures was considered for VLP based Influenza vaccine. In this experimental study، the human influenza virus (A /New Caledonia 20/1999/ (H1N1)) was propagated in MDCK cell culture. Viral RNA was extracted using RNX-plus solution. Complementary DNA synthesis was carried out using uni-12 primer and random hexamer as specific and general primers، respectively. Neuraminidase open reading frame (1413-bp) was amplified by PCR and cloned into pBlue-script SK. Neuraminidase coding frame sub cloned into pFastBac11 plasmid through SalI/XhoI sites. After verification of cloned Neuraminidase by restriction analysis، it was subjected to automated sequencing bi-directionally. The recombinant pFastBac Neuraminidase vector was transformed to E. coli DH10Bac cells which harbor bacmid DNA and helper plasmid to create Neuraminidase recombinant bacmid. Neuraminidase recombinant bacmid was created by homologous recombination between pFastBacNA and bacmid and was verified by PCR using Neuraminidase specific and M13 universal primers. Recombinant baculovirus expressing Neuraminidase gene can be also used with other individual recombinant baculoviruses expressing HA and M1 genes in production of influenza VLPs or proteins resulting from this structure could be purified in specific insects for vaccine research studies.