| Avian Influenza(AI) is a infectious disease caused by type A avian influenza viruses(AIV), which takes place in many countries and regions in the world, and causes great economic losses. What's worse, AI also threatens health of the public. Firstly, avian influenza viruses(AIV) can infect persons directly and cause death. Secondly, AIV can serve as the reservoir of genetic diversity permitting the emergence of pandemic influenza by means of genetic re-assortment with prevailing human influenza virus in the world. Vaccination is considered one of the most effective preventive measures for the control of influenza pandemics, so effective vaccines against H5N1 viruses are urgently needed. Now vaccination of HA and NA can only provide protection from lethal challenge with homologous influenza A viruses, furthermore, HA and NA can easily undergo antigenic drift or shift, no broadspectrum influenza vaccine has been developed till now. As a result, it is necessary to develop a broad-spectrum vaccine. In order to develop broad-spectrum vaccines providing cross-protection against different influenza variants, some recent studies have focused on influenza virus matrix protein 2(M2), which is a surface membrane protein of influenza A virus. M2 is one of the three integral membrane proteins, and has been remarkably conserved in all influenza A strains. M2 proteins form a proton-selective transmembrane ion channel and play a critical role in two stages of virus replication by regulating the pH of endosome and vesicular compartments of the trans-Golgi network(TGN). As a target antigen in various constructs, vaccination of M2 proteins can provide protection from lethal challenge with homologous and heterologous influenza A viruses in many studies. So M2 protein can be used as a universal influenza vaccine component. Owing to the protective epitope localizing on the extracellular domain of M2 protein(M2e), so now some studies have focused on M2e. But M2e is a hapten with only 24 aa, so it is needed to couple with a carrier to form a complete antigen. And in order to abtain large capacity, it is also needed to be expressed in a superior expression system. Here, the study was unfolded through the following aspects.1. According to the M2e peptides of H5N1 subtype AIV in GenBank, one 24 peptide was synthesized and conjugated to keyhole limpet hemocyanin(KLH). Then SPF chicken and BALB/c mice were immunized with the conjugates and equivalent adjuvant to prepare postive serum. In order to analyze the serum of immunized animals to identify IgG antibodies directed against M2, an indirect ELISA was established. in addition, its operation parameters were optimized. The optimal operation parameters were: 96-well microtiter plates were coated 1h at 37℃, then overnight at 4℃with 100μL 5μg/mL synthetic peptide. After washed by PBST, the plates were subsequently blocked 3h with 1% BSA,and then incubated for 45 min with 100μL sera diluted by 1% BSA. Then the plates were incubated with 100μL per well anti-chicken IgG immunoglobulin conjuate was added and incubated for 30 min at 37℃. After washing the plate, the indicator TMB was added and incubated 100μL per well for 15 min at 37℃. In the last, 50μLper well 2M H2SO4 was added, and the optical density(OD) was measured with a microtiter plate reader at 450 nm.2. According to the partiality codon of Pichia pastoris, influenza virus matrix 2 protein ectodomain segments(M2e) was amplified by SOE PCR using a couple of long primers. Then M2e gene and chicken IgG Fc segments gene were digested and inserted into Pichia pastoris expression vector pPICZaA, and the expression plasmid pPICZaA-M2-Fc was obtained. The recombinant plasmid was lineared and transformed into competant Pichia pastoris X-33 strain by electroporation. Then the recombinant transformants were selected by Zeocin and identified by PCR using the 3'AOX and 5'AOX primers. The expression of the fusion protein in yeast cells was induced by addition of methanol every 24 hours to 1%, and was analyzed by Tricine-SDS-PAGE and Western blotting. The results showed that the fusion expression vector for M2e and Fc segments was successfully constructed, and the relative molecular mass of the fusion protein was measured to be 23kDa. The fusion protein was expressed at a yield of 321 mg per liter of culture with a specific antigenicity as demonstrated by Western blotting.3. Eighteen chickens were divided into three groups and injected with the fusion protein M2e-Fc at the dosage of 150μg,100μg, 50μg respectively, while other three groups divided from 18 chickens were injected with KLH-M2e, supernatant of pPICZaA/X-33 and PBS as positive, negative and blank controls. Serum samples were obtained at week 0, 3, 5, 7, 11, 15 post-injection respectively and antibodies directed against M2e were determined by indirect ELISA. Then viral neutralization test were accomplished on SPF egg embryos and Madin-Darby canine kidney(MDCK) cell, at the same time, indirected fluorescence assay(IFA) was tested on MDCK infected by H9N2 viruses. At 7th week after immunization, T lymphocyte transformation(LT) rate, natural killer(NK) cell activity and macrophage activity were detected. The results showed that M2-specific antibodies of immunized groups were induced and can bind to M2 expressed on the surface of MDCK. Although M2-specific antibodies were not able to prevent virus adsorption and penetration in MDCK, they could inhibit the release of mature influenza virions. The results of cell-medicated immunity activity test suggested that immunization of M2e-Fc couldn't improve T lymphocyte transformation(LT) rate, natural killer(NK) cell activity and macrophage activity. In a word, immunization of M2e-Fc was able to induce the humoral immune response but not the cellar immune response.
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