Cloning and Expression of the Nucleoprotein Gene (NP) of Newcastle Disease Virus (NDV) in Escherichia coli for Immunodiagnosis Application

Silva, Ketherson Rodrigues; Gonçalves, M. C. M.; Oliveira, Érika Arantes de; Fernando, Filipe Santos; Montassier, Maria de Fatima Silv; Fernandes, Camila Cesário; Tamanine, Maria de Lourdes Fer; Borzi, Mariana Monezi; Santos, R. M.; Mendonça, André de Oliveira; Reischak, Dilmara; Paulillo, Antônio Carlos; Montassier, Hélio José

doi:10.3923/ijps.2014.473.479

Cited by 6 publications

(6 citation statements)

References 14 publications

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“…Although NPs of paramyxoviruses are relatively conserved, the monoclonal antibody reactivity could be detected antigenic differences among isolated NDV strains [ 8 , 26 ]. NP of negative-strand RNA virus possesses strong immunogenic in nature.…”

Section: Discussionmentioning

confidence: 99%

“…This may be explored as target to design NDV vaccines [ 7 ]. Latest studies have reported that the NP of NDV is an important antigen in serologic assays because of its highly conserved sequences and high immunogenicity [ 8 ]. However, the molecular evolution of NP gene in NDV and whether minor antigenic changes occur in this gene remain to be elucidated.…”

Section: Introductionmentioning

confidence: 99%

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Analysis of molecular evolution of nucleocapsid protein in Newcastle disease virus

Fan

Zhang

et al. 2017

Oncotarget

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The present study investigated the molecular evolution of nucleocapsid protein (NP) in different Newcastle disease virus (NDV) genotypes. The evolutionary timescale and rate were estimated using the Bayesian Markov chain Monte Carlo (MCMC) method. The p-distance, Bayesian skyline plot (BSP), and positively selected sites were also analyzed. The MCMC tree indicated that NDV diverged about 250 years ago with a rapid evolution rate (1.059 × 10−2 substitutions/site/year) and that different NDV genotypes formed three lineages. The p-distance results reflected the great genetic diversity of NDV. BSP analysis suggested that the effective population size of NDV has been increasing since 2000 and that the basic reproductive number (R0) of NDV ranged from 1.003 to 1.006. The abundance of negatively selected sites in the NP and the mean dN/dS value of 0.07 indicated that the NP of NDV may have undergone purifying selection. However, the predicted positively selected site at position 370 was located in the known effective epitopic region of the NP. In conclusion, although NDV evolved at a high rate and showed great genetic diversity, the structure and function of the NP had been well conserved. However, R0>1 suggests that NDV might have been causing an epidemic since the time of radiation.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Analysis of molecular evolution of nucleocapsid protein in Newcastle disease virus

Fan

Zhang

et al. 2017

Oncotarget

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“…Results of the experiment showed that the muti-epitopic recombinant HN and F antigens expressed in the E. coli could elicit a sufficient rise of antibody in the immunized mice for detection by ELISA as well as standard Newcastle vaccine which composed of the whole structural NDV proteins. On the other hand, the commercial strains that are currently being used for detection of NDV induce a high amount of antibodies which may not directly produce against HN or F antigens, because NDV structural proteins like nucleocapsid protein (NP) are also associated with the high immunogenicity properties, as well (17, 43).…”

Section: Discussionmentioning

confidence: 99%

Immunogenicity of the Multi-Epitopic Recombinant Glycoproteins of Newcastle Disease Virus: Implications for the Serodiagnosis Applications

et al. 2018

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Background Newcastle disease virus (NDV) is a dangerous viral disease, infecting a broad range of birds, and has a fatal effect on the poultry industries. The attachment and consequently fusion of the virus to the host cell membrane is directed by the two superficial glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) which is considered as the important targets for the poultry immune response. Objectives The principal goal of this investigation was to realize the potential efficacy of the E. coli expression system for the production of the multi-epitopic HN, and F proteins with respect to the ability for the stimulation of the immune system and production of the cross-reactive antibodies in mice. Materials and Methods The recombinant HN and F (rHN, rF) have accumulated almost 40% of the total bacterial proteins. The presence of rHN and rF proteins recognized by the Western blotting with specific anti-HN, anti-F, anti-Newcastle B1, and anti-poly 6x His-tag antibodies. Furthermore, both rHN and rF have shown the specific reactivity against the Newcastle B1 antiserum as a standard strain. Results The ELISA analysis showed that the higher dilutions of the antibody against Newcastle B1 could react with the as least quantity as 100 ng of the purified rHN, and rF. Cross-reactivity analysis of the sera from the mice immunized with Newcastle B1 in two time points indicated that the raise of anti-Newcastle B1, anti-HN and anti-F antibodies peaked at 28 days post immunization (dpi). Moreover, temporal variation in IgG titration between both time points was significant at 5% probability level. Conclusion The results provided valuable information about the cross-reactivity patterns and biological activity of the multi-epitopic proteins compared to the NDV standard strain which was determined by the Western blotting and ELISA.

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“…AIV-rNP-ELISA was performed according to the general protocol developed by Silva et al (2014). A checkerboard titration of four rNP concentrations (2, 4, 8, and 16 µg/ml) and six dilutions (1:50, 1:100, 1:200, 1:400, 1:800, and 1:1600) of the positive and negative serum controls was performed to determine the optimum antigen concentration and the ideal serum dilution.…”

Section: Indirect Elisa (Aiv-rnp-elisa)mentioning

confidence: 99%

Development and application of an enzyme-linked immunosorbent assay (ELISA) using a soluble recombinant nucleoprotein for the detection of antibodies to avian influenza virus

Borzi¹,

Silva²,

Montassier³

et al. 2017

Afr. J. Microbiol. Res.

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Avian influenza (AI) causes significant impact on industrial poultry farming, besides infecting a variety of vertebrates. The detection of antibodies against viral antigens by serological methods is important for the epidemiology, control and prevention of AI because their high simplicity and speed for assaying a large number of samples. Obtaining antigenic preparations used for detection of anti-avian influenza virus (AIV) antibodies usually requires complex and expensive procedures and Escherichia coli system expression may be an alternative. The nucleoprotein (NP) of AIV is an ideal antigen candidate because it is highly conserved across AIV strains, resulting in high cross-reactivity and immunogenicity for avian hosts. The NP gene segment was cloned and expressed from AIV isolate H4N6 in E. coli fused to a small ubiquitin-like modifier (SUMO) polypeptide and a poly-histidine tag, obtaining a soluble recombinant NP (rNP) containing the most important epitopes. After purification, the rNP was used as an antigen to develop an indirect rNP-enzyme-linked immunosorbent assay (ELISA) to effectively detect anti-AIV antibodies in chicken serum samples. This rNP-ELISA had high sensitivity (95%), specificity (97%), accuracy (96.7%) and agreement (k=0.88) in a comparative analysis with a commercial ELISA kit. The results suggest that rNP-ELISA offers a viable alternative to improve immunodiagnosis of AIV infection in chickens.

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Cloning and Expression of the Nucleoprotein Gene (NP) of Newcastle Disease Virus (NDV) in Escherichia coli for Immunodiagnosis Application

Cited by 6 publications

References 14 publications

Analysis of molecular evolution of nucleocapsid protein in Newcastle disease virus

Analysis of molecular evolution of nucleocapsid protein in Newcastle disease virus

Immunogenicity of the Multi-Epitopic Recombinant Glycoproteins of Newcastle Disease Virus: Implications for the Serodiagnosis Applications

Development and application of an enzyme-linked immunosorbent assay (ELISA) using a soluble recombinant nucleoprotein for the detection of antibodies to avian influenza virus

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