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Background: 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. Materials and Methods: 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. Results: Neuraminidase recombinant bacmid was created by homologous recombination between pFastBacNA and bacmid and was verified by PCR using Neuraminidase specific and M13 universal primers. Conclusion: 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.

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Background: Genetic variability of influenza viruses causes new epidemics worldwide annually. Development of a new vaccine for prophylaxis of influenza virus has been amajor objective in recent years. The aim of this study was to construct a recombinant baculoviruscapable of expressing the two surficial antigenic glycoproteins, hemagglutininand neuraminidase, as well as matrix proteinsof swine influenza (H1N1) simultaneously and independently. Materials and Methods: In this experimental study, first, a triplet cassette providing simultaneous and independent expression of target proteins was designed and subjected to synthesis. It was then cloned into pFastBac1 donor plasmid. Competent E.ColiDH10Bac cells were transformed by donor clone and the recombinant bacmids were produced following homologous transposition. This construction was verified by PCR and then transfected into Sf9 insect cells to package new recombinant baculoviruses. Results: Restriction map of pFastBacI HNM1 donor plasmid confirmed the fidelity of the clone. The results of PCR done on the recombinant bacmidas template indicated that a proper homologous recombination has occurred between pFastBacI HNM1 donor plasmid and the bacmid in E.ColiDH10Bac host cells. Protein analysis of the infected Sf9 cells showed that all target proteins were efficiently expressed at the same time. Conclusion: The recombinant baculovirus constructed in this studypossesses proper characteristics to produce swine influenza virus-like particles in Sf9 cells.

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Background: Newcastle disease virus (NDV) is one of the major pathogens in poultry and vaccination is intended to control the disease, as an effective solution, yet. Fusion protein (F) on surface of NDV, has a fundamental role in virus pathogenicity and can induce protective immunity, alone. With this background, here our aim was to construct a baculovirus derived recombinant bacmid shuttle vector (encoding F-protein) in order to express it in insect cell line.

Materials and Methods: In this experimental study, at first complete F gene from avirulent strain La Sota of NDV was amplified by RT-PCR to produce F cDNA. The amplicon was cloned into T/A cloning vector and afterwards into pFastBac Dual donor plasmid. After the verification of cloning process by two methods, PCR and enzymatic digestion analysis, the accuracy of F gene sequence was confirmed by sequencing. Finally, F-containing recombinant bacmid was subsequently generated in DH10Bac cell and the construct production was confirmed by a special PCR panel, using F specific primers and M13 universal primers.

Results: Analysis of confirmatory tests showed that the recombinant bacmid, expressing of F-protein gene in correct sequence and framework, has been constructed successfully.

Conclusion: The product of this F-containing recombinant bacmid, in addition to its independent application in the induction of protective immunity, can be used with the other individual recombinant baculoviruses, expressing HN and NP genes to produce NDV-VLPs in insect cell line.