Isolation And Identification Of H1n1 Subtype Of Siv And Development Of Diagnostic Method And Immunogenicity Of M2e Recombinant Plasmid

Swine influenza(SI) is a acute contagious infectious respiratory disease caused by swine influenza virus(SIV). Clinical signs include high fever, labored breathing, abortions, coughing, snivel and high percentage of morbidity and a low percentage of deaths. SI which don't cause large scale swine dead often occur in globe, through weaken health and product performance of pig. Pigs not only be delay to be listed, but also be influence their economic benefit. It is commonly known that SI play a very important role in Porcine respiratory disease complex (PRDC), which can't be ignore even inferior to PRRSV and CFSV. Efficient and accurate detection technology and safe and reliable vaccine is very essential to prevent and control SIV and also is hotspot in research. This research was aim to isolate and characterize a H1N1 subtype SIV and construct a indirect ELISA method which can distinguish street strain infection and inactivated vaccine inoculation. Base on self-conatructing polypeptide which include three copy of M2e and researching its related biological activity, we research immunogenicity of M2e recombinant plasmid with adjuvant of swine IL-18 and PRV VP22, and the protection effect of combine HA and M2e.1. Isolation and characterization of H1N1 subtype swine influenza virusA H1N1 subtype SIV A/Swine/Sichuan/37/209 (H1N1) was separated by using MDCK cells and embryonated egg, and its biological characteristics were studied. By seq uencing analysis of whole gene sequence of HA and NA, we found this strain has high homology with H1N1 subtype influenza of human-origin (A/Gunma/07G002/2008 and A/G uangzhou/665/2006),and nucleotide homology respectively was 99.4% and 99.7%。The n ucleotide substitutions tree also proved that A/Swine/Sichuan/37/2009 (H1N1) may be human-origin.The RT-PCR based on consertive sequence of NS gene of influenza A vir us which is well sensitive and special and preliminary could detect swine influenza virus quickly and accurately in clinical.2. Construct of Indirsect-ELISA of NS1 protein of SIVThrough cloning NS1 gene of swine influenza A/Swine/Sichuan/01/2006(H3N2) and sequnening it, we found among different subtype strains have high homology and six re latively conservative antigen determinant. The prokaryotic expression which was constructe d by NS1 gene and pET32-a(+) was induced by IPTG, and the fusion protein expressed was 43.5KD after detected by SDS-PAGE electrophoresis. The fusion protein could speci ally react with positive serum of SIV after detected by Western-blotting and be the coate d antigen of Indirect-ELISA which has relative good speciality and sensitivity and could distinguish street strain infection and inactivated vaccine inoculation. It is practicable that developing a kit base on NS1 protein.3. Prokaryotic expression,eukaryotic expression and biological activity researching of M2eThe prokaryotic expression and eukaryotic expression plasmid of M2e gene constructe d three copies of M2e gene using over-lapping PCR. The prokaryotic expression fusion p rotein which was 29KD could react with positive serum of H1N1 and H3N2 subtype inf luenza by Western-blotting and immunized mouse to produce high titer antibody (GMT=1 4336)which can recognize antigen on the membrane of influenza virus infected cell.The a ntibody couldn't neutralize virus in vitro but could show passive immunity in vivo to pre vent mouse to lethal dose homologous and heterologous virus challenging. The eukaryotic expression plasmid could efficient express in cell in vitro so that provide a good founda tion to researching immunogenicity of M2e recombinant plasmid.4. Preliminary study in Swine Interleukin 18 (IL-18) and VP22 of PRV using as molecular adjuvantThe eukaryotic expression plasmid of IL-18 could express IL-18 protein (400.6±21.88μg/L) and active potential of mouse spleen Lymphocyte proliferation (SI=3.016±0.244).The VP22 gene of PRV(Fa strain) after sequenced was found high homology with Kaplan strain and Ea strain.Constructing a eukaryotic expression plasmid by inserting VP22 into upstream of EGFP gene of pEGFP-N3 plasmid, the plasmid could transcripe and translate in vitro. The plasmid pVP22-HA construct expressing a fusion protein of VP22 joined to the N terminus of the HA induced mouse producing more higher titer antibody response of HI (230.4±57.24) than sole pCI-HA (102.4±35.05).5. M2e recombinant plasmid and influence of IL-18 and PRV VP22 to immunogenicity of M2e recombinant plasmid We respectively constructed eukaryotic expression plasmid of pCI-M2e*3, pM2e*3-IRES-IL18 and pVP22-M2e*3, and proved them can transcripe and translate by RT-PCR and indirect immunofluorescence. The three plasmid could induced mouse producing different degree of humoral inmunity and cellular immunity. IL-18 and VP22 played role as adjuvant to M2e because that many index of pM2e*3-IRES-IL18 and pVP22-M2e*3 surpassed pCI-M2e*3. After challenged by lethal dose virus, three immunized-groups showed a undesirable protect effectiveness according to body weight change and survival rate that is pVP22-M2e*330% and the another 10%; pVP22-M2e*3 didn't show any less mild lung lesions compare to control group. pVP22-M2e*3 show some degree of heterologous protecting capacity.6. The researching of immunogenicity of M2e & HA fusion expressing recombinant plasmidThe plasmid pM2e*3-HA construct expressing a fusion protein of M2e joined to the N terminus of the HA could transcripe and translate. The special antibody of M2e of mouse immunized pM2e*3-HA (384.8±134.92) show remarkable higher titer than pCI-M2e*3 (24±8.43) immunized group, and almost no difference to pCI-HA immunized group in HI antibody but a slightly higher neutralization antibody but not significant. After challenged by lethal dose virus, pM2e*3-HA immunized group show a satisfied effectiveness according to body weight change and survival rate(100%) and own a good heterologous protecting capacity; After challenged by moderate dose virus, pM2e*3-HA group could alleviate lesion of lung compare to control group. It is that fusion HA and M2e provied a new method and ideal to research-develop a new type SI vaccine.