Characterisation of an avian influenza virus nucleoprotein expressed inE. coli and in insect cells (original) (raw)
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Virus Research, 1985
The influenza virus nucleoprotejn gene has been cloned by a procedure that involves direct cDNA synthesis onto the primer-vector pBSV9, a pBR322-SV40 recombinant plasmid. dT-tailed pBSV9 was used to prime the synthesis of cDNA on a template of in vitro synthesized viral mRNA. The synthesis of ds-cDNA was initiated by a specific oligodeoxynucleotide and the resulting recombinant was circula~zed by intramolecular ligation. Recombinant pSVa963 contained the viral nucleoprotein gene directly fused to the SV40 early promoter region included in pBSV9 and followed by a dA : dT tail and the SV40 polyadenylation signal. When pSVa963 was used to transfect COS-1 cells, the presence of three NP-specific mRNAs of 1600, 1900 and 2500 nucleotides in length could be detected. Pulse labelling experiments of COS-1 transfected cells and immunobinding to a nucleoprotein monoclonal antibody indicated the synthesis of nucleoprotein. This nucleoprotein accumulated in the nucleus of transfected cells at a level similar to that found in infected cells. The vector and method described may be useful for the specific cloning and expression of any mRNA for which a 5'-terminal sequence is known. mRNA cloning, gene expression, DNA transfect~on, specific priming
Iranian Journal of Virology
Background and Aims: Influenza virus nucleoprotein (NP) has the capacity to be used as subunit vaccine, but little is known about the impact of different cultures on its structure. In the present study we aimed to evaluate and compare the Isoelectric focusing (IEF) property of extracted viral nucleoproteins derived from Madin Darby canine kidney (MDCK) cell line and embryonated chicken eggs (ECE). Materials and Methods: Influenza virus strain A/NewCaledonia/20/99/H1N1 was propagated and grown in allantoic sac of 10-11 day-old embryonated chicken eggs, and mammalian cell culture (MDCK) in parallel. Ribonucleoprotein extraction was conducted from two separate cultures and evaluated using isoelectric focusing gel strips. Results: The results showed higher isoelectric pH in extracted nucleoproteins from MDCK as compared to embryonated chicken eggs. Conclusion: It is possible that some amino acids have been replaced. Suggesting that the changing net charge of protein may be affect the conserved regions of the protein. Therefore, this could impact the new generation of vaccines construction based on conserved proteins.
Monoclonal antibodies in immunodiagnosis and immunotherapy, 2013
The present study was carried out with an aim to develop anti-nucleoprotein (anti-NP) monoclonal antibodies (MAbs) for use in immunodiagnostic testing for detection of avian influenza virus (AIV) antigen or antibodies. The NP gene of AIV, cloned in pET vector, was expressed in Escherichia coli BL 21 strain to produce a 6x-His tagged recombinant NP (rNP) antigen of ∼61 kDa molecular weight as soluble fraction. The rNP antigen was detected in soluble fraction of bacterial cell lysate with anti-His HRPO conjugate and reacted with the reference AIV antibody positive serum in immunoblotting. The rNP was used to immunize BALB/c mice to produce hybridoma secreting anti-NP MAbs. Out of 11 anti-NP MAbs produced, 8D2-H5, 8D2-H9, and 6D11-A7 were of IgM isotype and 5D10-C9 and 5D10-F11 were of IgG2b type, while 3F3-D2, 7D2-C9, 7D2-G7, and 7D2-G8 were of IgG1 isotype. The MAbs 3F3-D2 and 7D2-G8 showed high intensity positive reaction with rNP and a low intensity reaction with H5N1 virus in West...
Functional expression of influenza A viral nucleoprotein in cells transformed with cloned DNA
Virology, 1986
Simian cells permissive for influenza A virus infection were stably transformed with a full-length cloned influenza A nucleoprotein gene under the control of an inducible metallothionein promoter and linked to a dihydrofolate reductase gene to facilitate cell selection. Transformed cells synthesized a virus-specific nucleoprotein which was indistinguishable from the nucleoprotein synthesized in virus-infected cells with respect to molecular weight and intracellular localization. It was estimated that transformed cells produced only 1% of the amount of nucleoprotein synthesized in simian cells infected with influenza A virus. Nontheless, when transformed cells were infected with influenza virus mutants which synthesized temperature-sensitive nucleoprotein, protein expressed by the cloned gene was able to complement the synthesis of plus-strand and minus-strand viral RNA for one mutant and only plus-strand synthesis for another mutant. This indicated that the influenza A nucleoprotein expressed in the transformed cells exhibited functional activity.
The Journal of general virology, 2000
A systematic analysis was carried out to identify the amino acid signals that regulate the nucleo-cytoplasmic transport of the influenza A virus nucleoprotein (NP). The analysis involved determining the intracellular localization of eight deleted recombinant NP proteins and 14 chimeric proteins containing the green fluorescent protein fused to different NP fragments. In addition, the subcellular distribution of NP derivatives that contained specific substitutions at serine-3, which is the major phosphorylation site of the A/Victoria/3/75 NP, were analysed. From the results obtained, it is concluded that the NP contains three signals involved in nuclear accumulation and two regions that cause cytoplasmic accumulation of the fusion proteins. One of the karyophilic signals was located at the N terminus of the protein, and the data obtained suggest that the functionality of this signal can be modified by phosphorylation at serine-3. These findings are discussed in the context of the tra...
Expression of influenza neuraminidase in baculovirus-infected cells
Virus Research, 1992
Recombinant influenza neuraminidase (NA, subtype 2, A/NT/60/68) was produced by recombinant baculovirus-infected insect cells. The recombinant NA retained enzyme activity and was located on the cell surface. Enzyme activity was both cell-associated and in the cell free supernatant; maximal NA activity was found in the supernatant. Recombinant NA was recognised by polyclonal antisera and by three monoclonal antibodies specific for NA (subtype 2). Enzyme activity was also neutralised by polyclonal antisera. Recombinant NA thus retains most of the immunological and activity properties of authentic influenza NA. Immunoprecipitation of [ 35S]Methionine-labelled cells and supernatant and partial purification of NA indicated that a N 50-kDa form of NA was present in the supernatant, whilst the expected size ( N 67-kDa) was cell-associated. Purified recombinant extracellular virus was also enzymatically active, and contained the 67-kDa NA which was located on the membrane capsule of the virus. This suggests that the virus had acquired the cell-associated form of recombinant NA during the budding process from the cell.
Cloning and Expression of Recombinant Nucleoprotein of Influenza H1N1
Novelty in Biomedicine, 2015
Background: Influenza virus is the major cause of lower respiratory tract illnesses on the worldwide. Vaccination can be an effective tool to prevent its outbreak. Highly conserved viral nucleoprotein is an effective vaccine candidate to provide heterosubtypic immunity, offering resistance against various influenza virus strains. Materials and Methods: In present research NP gene was inserted in pET-22b expression vector. New construct (pET-22b/NP) was transformed into E. coli BL21 (DE3) strain and the expression of nucleoprotein was induced by IPTG. It was analyzed by SDS-PAGE and confirmed by Western blotting. Results: Western blotting confirmed the expression and production of recombinant Influenza nucleoprotein. Conclusion: These results suggest that the codon-optimized influenza A virus NP gene can be efficiently expressed in E. coli.
Journal of Virology, 1995
The influenza A virus nucleoprotein (NP) has been examined with regard to its RNA-binding characteristics. NP, purified from virions and devoid of RNA, bound synthetic RNAs in vitro and interacted with the ribonucleotide homopolymers poly(A), poly(G), poly(U), and poly(C) in a salt-dependent manner, showing higher binding affinity for polypyrimidine homopolymers. To map the NP regions involved in RNA binding, a series of deleted forms of the NP were prepared, and these truncated polypeptides were tested for their ability to bind poly(U) and poly(C) homopolymers linked to agarose beads. Proteins containing deletions at the N terminus of the NP molecule showed reduced RNA-binding activity, indicating that this part of the protein was required to bind RNA. To identify the NP region or regions which directly interact with RNA, proteins having the maltose-binding protein fused with various NP fragments were obtained and tested for binding to radioactively labeled RNAs in three different ...