Abstract
As an attempt to increase the resistance to Newcastle Disease Virus (NDV) and so further reduction of its risk on the poultry industry. This work aimed to build the eukaryotic gene co-expression plasmid of neuraminidase (NA) gene and myxo-virus resistance (Mx) and detect the gene expression in transfected mouse fibroblasts (NIH-3T3) cells, it is most important to investigate the influence of the recombinant plasmid on the chicken embryonic fibroblasts (CEF) cells. cDNA fragment of NA and mutant Mx gene were derived from pcDNA3.0-NA and pcDNA3.0-Mx plasmid via PCR, respectively, then NA and Mx cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to generate the eukaryotic co-expression plasmid pVITRO2-Mx-NA. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequencing, and it was transfected into the mouse fibroblasts (NIH-3T3) cells. The expression of genes in pVITRO2-Mx-NA were measured by RT-PCR and indirect immunofluorescence assay (IFA). The recombinant plasmid was transfected into CEF cells then RT-PCR and the micro-cell inhibition tests were used to test the antiviral activity for NDV. Our results showed that co-expression vector pVITRO2-Mx-NA was constructed successfully; the expression of Mx and NA could be detected in both NIH-3T3 and CEF cells. The recombinant proteins of Mx and NA protect CEF cells from NDV infection until after 72 h of incubation but the individually mutagenic Mx protein or NA protein protects CEF cells from NDV infection till 48 h post-infection, and co-transfection group decreased significantly NDV infection compared with single-gene transfection group (P<0. 05), indicating that Mx-NA jointing contributed to delaying the infection of NDV in single-cell level and the co-transfection of the jointed genes was more powerful than single one due to their synergistic effects.
Highlights
Avian influenza viruses (AIV) are enveloped, segmented and negative-stranded RNA viruses, which circulate in world-wide and have caused the biggest concern bird flu and severe poultry industry economic losses [1]
Expression of Chicken myxo-virus resistance (Mx) and NA mRNA in NIH 3T3 Cells To confirm the expression of the chicken Asn631 Mx mRNA or NA in eukaryotic cells, the expression vector pVITRO2-Mx, pVITRO2-NA and pVITRO2-Mx-NA was transfected into chicken embryonic fibroblasts (CEF) cells
It was reported that molecular biology function of NA protein is to remove the virus particles and sialic acid, which is important for the release of viral particles and prevent the aggregation of the virus particles [17]
Summary
Avian influenza viruses (AIV) are enveloped, segmented and negative-stranded RNA viruses, which circulate in world-wide and have caused the biggest concern bird flu and severe poultry industry economic losses [1]. NA gene encoding the neuraminidase protein that is a target antigen of humoral immune, which could induce specific antibodies and inhibit virus release from the infected cells, thereby reduce the virus proliferation and increase the immune protection function [2]. Sialic acid on the host cell membrane is the main receptor of influenza and NDV. These viruses only bind with that receptor causing lesions on the host infected cell. NA can degrade the sialic acid receptor and protect the cells from AIV and NDV infection [3]. NA has become the active research field due to its role against influenza virus
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