Expression And Anti-virus And DNA Vaccine Immune Activity Of IFN Alpha And Gamma From Jilin White Goose

Interferon has broad-spectrum antiviral activity and effective immunoregulation. So it has important significance to develop effective antiviral biological agent and enhanced vaccine adjuvant.JGIFN-α/γ has been cloned, expressed studied antiviral activity in this study. Further study on JGIFN-γ of immunological enhancement.Carrying molecular immune adjuvant DNA vaccine has been constructed and immuned to gosling.The study laid a foundation for goose parvovirus DNA vaccine.the study included as follows:1. The research of prokaryotic expression and antiviral activity of mJGIFN-aThe mature peptide gene was amplified by one pair specificity primer based on the cloned. Then the mJGIFN-a was cloned into pET-28a(+) vector and constructed prokaryotic expression plasmid pET-mJGIFN-a.The22ku expressed protein was obtained Ni-IMAC after affinity chromatography and dilution dialysis renaturation. The anti-VSV of recombinant protein mJGIFN-a activity by micro cytopathogenic effect inhibition assay was1.3×103U/mg.It was detected the anti-GPV of recombinant protein by FQ-PCR.The resuts showed the antivity of mJGIFN-a to GPV protein was detected.2. Sequence analysis and expression and antivirus activity detection of JGIFN-γThe Jilin white goose interferon-gamma (JG-IFN-γ) was amplified from ConA-stimulated peripheral blood mononuclear cells (PBMCs) by RT-PCR. Then constructed the recombinant plasmid (pMD-JGINF-γ) sucessfully. The nucleotide and the deduced amino acid sequence of JGINF-γ were analyzed by bioinformatics software and on-line tools. The homology with13other correlative gene sequences was analyzed. The homology showed that the JGINF-y has more than90%similarity to waterfowl (duck and goose) and in the same branch in the phylogenetic tree. It has the most homology with dometic goose of which99.6%nucleotide and97.6%amino acid. The protein wasl9ku.The results analysise showed three N-glycosylation sites, ten potential phosphorylation sites, four potential hydrophobic regions were in the JGIFN-γ amino acid.The analysis of the protein subcellular location indicated that the JGIFN-γ mostly located at nuclear, others located at mitochondrial, cytoplasmic, plasma membrane, vesicles of secretory system, extracellular,including cell wall and Golgi.The secondary structure prediction results showed that JGIFN-y was composed of a-helix with four epitopes of higher antigen index. Then JGIFN-y was constructed prokaryotic expression plasmid (pET-JGIFN-γ) and expressed in strain rossetta. The22ku protein was obtained after Ni-IMAC affinity chromatography and dilution dialysis renaturation.The anti-VSV activity of recombinant protein by micro cytopathogenic effect inhibition assay was4.3×102U/mg. 3. The study of eukaryotic expression and preparation of JGIFN-γ/JGIFN-γ-VP3DNA gene vaccineJGIFN-γ-VP3fusion gene which lacked TAA stop codon of JGIFN-γ and ATG iniation codon of VP3gene and linked by high hydrophilia amino acid (Gly-Gly-Gly-Ser) encoding nucleotides was amplied by using spicing by overlap extention. Then JGIFN-γ-VP3fusion gene and JGIFN-γ was inserted into pVAX1under the control of CMV respectively. pVAX-JGIFN-γ and pVAX-JGIFN-γ-VP3were constructed successfully,which were then transfected into the vero cells by lipofection transfection.4. The immunity study of eukaryotic expression plasid JGIFN-γ-VP3in the goslingsIn order to study on the immunity fuction of pVAX-JGIFN-γ and p VAX-JGIFN-γ-VP3. The pVAX-JGIFN-γ-VP3of the different doses and the pVAX-JGIFN-γ of the different doses with pVAX-VP3DNA vaccines was immunized to28-day old geese. The VP3DNA vaccine,the empty vector plasmid pVAX1, Gosling Plague Attenuated vaccine and saline was served as control group. The anticoagulated blood samples were harvested at different times to detect the proliferation of geese T lymphocytes in PBMCs by T lymphocyte proliferation tests(MTT).The results showed that the pVAX-JGIFN-γ-VP3immunized groups were higher than the same dose of pVAX-JGIFN-y+pVAX-VP3immunized groups. The adjuvant groups significantly higher than (P<0.01) higher than pVAX-VP3DNA vaccine group,but lower than GPV attenuated groups. The kinetics of IgG of serum samples which were collect from immunized geese was detected by indirect ELISA. The results showed that ELISA antibody of humoral immunity induced by pVAX-JGIFNy-VP3and pVAX-JGIFNy+pVAX-VP3was higher than pVAX-VP3DNA vaccine. The GPV attenuated groups were higher than all of DNA vaccine. The neutralizing antibody level of pVAX-JGIFNy-VP3and pVAX-JGIFNy+pVAX-VP3immuized was detected by virus neutralization test. The results showed neutralizing antibody of all of vaccines was produced.the adjuvant of GPV-VP3DNA vaccines were significant higher than GPV-VP3DNA vaccine. The neutralizing antibody level induced by three kinds of GPV DNA vaccines and GPV attenuated vaccine maintained longer times. but the adjuvant DNA vaccine groups was higher than DNA vaccine groups.The neutralizing antibody induced by all of vaccines can protect GEF from infecting GPV to some extent...