| Avian influenza, caused by avian influenza virus (AIV), is a highly contiguous disease. AIV can infect almost all wild and domestic poultries and cause severe economic losses for the poultry industry. The M2 protein of AIV is a transmembrane protein. It has highly conserved antigenic epitopes and is a potential candidate antigen for avain influenza vaccine with cross-protection.In this study, M2 gene of AIV was cloned and its transmembrane segment was deleted and prokaryotically expressed. The influence of amantadine on M2 expression was analyzed. Monoclonal antibodies (McAbs) against M2 were prepared using the fusion protein as an immunogen. The M2 gene labeled with green fluorescent protein (EGFP) was inserted into the US gene which is a nonessential region of genome of MDV CVI988 and an EGFP marked recombinant plasmid was obtained. By homologous recombination technology, a recombinant Marek's disease virus expressing AIV M2 gene was successfully constructed. The protective effect of the recombinant virus against AIV was primarily studied.M2 gene of AIV A /chicken /Guangdong/2000 (H9N2) was amplified by RT-PCR and the amino acids (27-49aa) of its transmembrane segment was deleted using overlapping PCR (OE-PCR). Then the recombinants containing the full-length M2 or the M2 deleted transmembrane segment were constructed using prokaryotic expression vector pGEX-5x-l. The recombinant plasmid was transformed into E. coli (BL21/DE3), induced and the expression of the two genes was measured by SDS-PAGE and Western blot. The expressed proteins were purified with glutathione sepharose 4B column by affinity chromatography. Immunofiuorescence test was conducted by using sera prepared in mice agaist purified M2 protein and AIV (H5N1) infected MDCK cells. The results showed that the M2 deleted transmembrane segment was highly expressed with an anmount of 22% and it was a fusion protein in a soluble form. The western blot analysis showed that the recombinant protein could specifically react with sera from SPF chicken immunized with AIV H5N1 virus strain, and the antisera to M2 protein could bind with AIV(H5N1)infected MDCK cells. These results suggested that the recombinant protein shared good immunogenicity.Amantadine is known as the specific inhibitor for the toxicity of the ATV M2 protein. In this study, the recombinants containing full length M2 and the M2 deleted transmembrane segment were expressed in E.coli. BL21 (DE3 ) respectively. While IPTG was added to the cell culture, various concentration of amantadine was also tested. The results showed that the recombinant containing full length M2 failed to express, even in the presence of different concentration of amantadine, while the recombinant containing the M2 deleted transmembrane segment was highly expressed in E.coli. BL21 (DE3) . However, the toxicity of the recombinant protein to the host cell still existed and couldn't be inhibited by amantidine.Purified fusion protein was used to immunize Balb/c mice for generating hybridoma cell lines The hybridomas showing strong positive reaction to M2 fusion protein and negative to GST were selected using M2 fusion protein and GST as ELISA antigen. Four positive clones against M2 protein were obtained and labeled as 1E1, 2F8, 4E3 and 5D2 respectively. These McAbs presented high ELISA titers and agar diffusion titers. They could react specifically with A1V H9 and H5, but not react with Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV). The IFA and immunohistochemical test confirmed that the McAbs could specifically bind to AIV in the MDCK cells, chicken embryo fibroblast cells and chicken tissues.The M2 gene ligated with green fluorescent protein (EGFP) was inserted into the US gene, which located in the nonessential region of MDV1 (CVI988) genome and an EGFP marked recombinant plasmid was obtained. Through homologous recombination, this construct was transfected into cells infected with Marek's disease virus CVI 988/Rispens. PCR test, dot-blot and western-blot results demonstrated... |
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