Studies On The Antiviral Activities And The Immunological Adjuvants Of Porcine Interferon-alpha, Interferon-beta & Porcine Interferon-gamma

Interferon(IFN) is a kind of cytokine with antiviral activities, anti-tumor and regulatory immunity functions. Since Isaacs and Lindenmann reported in 1957, the studies on IFN and its development and application never stopped. Now it has been utilized in a variable of disseases of human being. In 1980, IFN was classified into three categories basing on the antigenic specificity by world health organization(WHO), IFN-α, IFN-βand IFN-γ. Afterwards it was classified into two types on the different acceptors, typeâ… IFN and typeâ…¡IFN. Typeâ… IFN includes IFN-α,β, w,Ï„,δetc, and typeâ…¡IFN only contains IFN-γ.Relatively speaking, the studies on the animal IFN began comparatively late in veterinary medical circles. In pork-producing community, various infectious diseases which are caused by viral or bacterial pathogens, badly restricted the development of natural and regional farming industries, and also seriously affect human health in whole world. Hence, it's necessary and great meaningful in both economic development and societal stabilization to prevent and control infectious diseases. In light of the consideration, in this study, porcine interferon-alpha(PoIFN-α), porcine interferon-beta(PoIFN-β) and porcine interferon-gamma(PoIFN-γ) were investigated for their antiviral effects on PRV, PRRSV and FMDV and their adjuvant effects on routine vaccine(c-strain, CSFV) with the form of recombinant proteins or the DNA form. The main researches included as below:1. Cloning and sequence analysis of porcine interferon gamma(PoIFNγ) gene and its expression in E.coliThe total RNA was extracted from peripheral blood mononuclear cells(PBMC) which was isolated from Meishan porcine and induced with concanavaline A(ConA), then the porcine interferon gamma(PoIFNγ, 501bp) gene was amplified via RT-PCR. The result of sequencing demonstrated that the amplified PoIFNγhad 100% nucleotide homology of other porcine IFNγsequence published on Genbank. And the amplified PoIFNγhad 78%, 77%-80%, 79%-80%, 77%, 71%, 60%, 59%-60%, 43% of amino acid sequence homology compared with caprine, cervus, camel, cattle, horse, rabbit, human, murine. PoIFN-γgene was inserted into pGEX-KG, and the recombinant plasmid was transformed into BL21 E.coli strain, which was induced by IPTG and expressed the interest protein. The expressed protein was proven to be the fusion protein of PoIFN-γand GST by SDS-PAGE. And the protein(43 kDa) was mainly insolvable inclusion body, which was about 69.91% of the total host bacterium. And the solvable protein was about 15.64% of the total host bacterium. The best inducing time was 5 h after the addition of IPTG and the concentration of the interest protein was 0.86 mg/mL. The inclusion body of PoIFN-γafter degeneration, renaturation, dialysis and purification was added to Madin-Darby bovine kidney cells(MDBK) to test the anti-VSV activity. And the results indicated that the expressed protein had highly biological activity, which as high as to 1.0×l0⁵U/mg.2. Secreting expression of porcine interferon-alpha in Pichia pastoris and the inhibition of PoIFN-βto FMDVTo highly express secreted porcine interferon-alpha/beta/gamma(PoIFNα/β/γ), the signal peptides were respectively excised from the PoIFNα/β/γgenes and the mature genes(mPoIFNα/β/γ, 501/498/435 bp) were respectively cloned into the yeast-Escherichia shuttle vector pPIC 9K to construct secreting recombinant expressing plasmid of pPIC 9K-mPoIFNα/β/γ. The pPIC 9K-mPoIFNα/β/γwere linearized by Salâ… and co-transformed with ssDNA into Pichia pastoris cells GS115(defective with histidine) with LiCl. The transformants were selected with MD culture plate and identified by PCR. And then, the multicopy recombinant Pichia pastoris strain was selected by G418 resistance. The selected strain could specifically secret mPoIFNα/β/γproteins which were demonstrated by SDS-PAGE and Western blot. The results of physicochemical properties and the antiviral activities of the secreted protein indicated that the recombinant proteins had highly antiviral activities. Besides, the expressed mPoIFNβcould suppress FMDV in IBRS-2 cells.3. The eukaryotic expressions of PoIFN-α/β/γin different cell lines and the comparison of the effects of the single type PoIFN or the combination of typeâ… with typeâ…¡PoIFN on the replication of pseudorabies virus(PRV)Three eukaryotic expression plasmids respectively containing PoIFN-α/β/γwere constructed. And the pcD-PoIFNα/β/γwere respectively transfected into HeLa,MDBK,BHK-21,PK-15 and IBRS-2 cell lines. The culture supernatants were assayed by anti-VSV(vesicular stomatitis virus) activity at 6, 24, 48 and 72 h post-transfection(hpt). Of the five cell lines analyzed, it was found that the expressing production reached the peak at 48 hpt. And PK-15, IBRS-2 and BHK-21 transfected with pcD-PoIFNαhad significant higher titer(256～890 times) of transient expression than MDBK and HeLa cell lines. IBRS-2 and BHK-21 transfected with pcD-PoIFNβhad higher titer(2.2～6times) of transient expression than PK-15 and HeLa, and PK-15 and HeLa were significantly higher(10～14 times) than MDBK. As for cells transfected with pcD-PoIFNγ, there was no significant difference in the titer among PK-15, IBRS-2, BHK-21 and MDBK.In this study, we compared the capacity of porcine IFN-α,-βand-γand the combination use of them to protect IBRS-2 cells from infection with pseudorabies virus (PRV). The results demonstrated that porcine IFN-β(PoIFN-β) was the most efficient in the three IFNs to resist PRV, which inhibited PRV plaque formation by 5.3-fold with 100U/mL PoIFN-β. Compared with PoIFN-β, porcine IFN-γ(PoIFN-γ) had less capability of inhibiting PRV plaque formation by 3.3-fold. While porcine IFN-α(PoIFN-α) had the least capability in the three PoIFNs, which inhibited PRV plaque formation by 1.26-fold and the inhibiting capability only increased to 2.3-fold with the treatment of 12800U/mL PoIFN-α. Furthermore, the combination of PoIFN-γand PoIFN-αor PoIFN-βinhibited PRV plaque formation by 12.8-fold or 100-fold, respectively. Treatment of IBRS-2 cells with PoIFN-α/βand PoIFN-γinhibited PRV replication by 29-or 146-fold. Additionally, real-time PCR analyses of the PRV immediate early(IE) gene revealed that IE mRNA expression was profoundly decreased in cells stimulated with PoIFN-α/βand PoIFN-γ(23.8-133.0-fold) as compared to vehicle-treated cells. The results indicated that PoIFN-γcould synergize with PoIFNs(PoIFN-αand-β) to potently inhibit PRV in vitro.4. Expressions of PoIFN-α/β/γmediated by recombinant adenoviruses(rAd) and the anti-PRRSV effects of rAd-PoIFNαPoIFNα/β/γgenes were sub-cloned via PCR and inserted into adenoviral shuttle vector, pShuttle-CMV, to construct recombinant plasmid pSh-PoIFNα/β/γ. pSh-PoIFNα/β/γwas linearized with Pmeâ… and dephosphorylated, then they were co-electrotransformated with adenoviral skeletal vector pAdEasy-1 into competent cells of BJ5183. The transforms were cultured at 37°for 24h on kanamycin resistance plate and selected for small colonies. Then, the extracted recombinant plasmids were named as pAd-Sh-PoIFNα/β/γ, which were conformed by Pacâ… digestion, and transformed into XL10-Glod(?) for copious preparation. pAd-Sh-PoIFNα/β/γlinearized with Pacâ… were co-transfected with liposome into 293 package cell line. After 7d-10d, the typical cytopathic effect indicated that recombinant adenoviral genome(deleted with E1 and E3 genes) carrying PoIFNα/β/γwere successfully packaged into intact virion. The recombinant virions were successive seeded to the several generations and the viral genomes were extracted from each generation for PCR. The PCR results demonstrated that the recombinant adenovirus carrying PoIFNα/β/γcould be stably passaged. By using cytopathic effect inhibition assay, the supernatant of the selected 293 A was tested for the antiviral bioactivity in bovine kidney cells(MDBK) and the anti-VSV(vesicular stomatitis virus) activities of rAd-PoIFNα/β/γwere 83640320U/mL, 5242880U/mL, and 1310720U/mL, respectively. The results indicated that the recombinant adenovirus successfully transformed PoIFNα/β/γand expressed PoIFNα/β/γwith good biological activities.MARC-145 cells were incubated with 100, 400, and 327680U/mL PoIFN-αfrom 293A cells infected with Ad5-PoIFNα/β/γor control supernatant fluids from 293A cells infected with an Ad5 control virus(Ad5-null). After 20 h, treated cells were infected with PRRSV of different TCID₅₀; MARC-145 cells incubated with Ad5-null supernatants developed cytopathic effects(CPE), whereas those incubated with high dose PoIFN-αshowed no CPE with dose-dependent and time-dependent. Additionally, real-time PCR analyses of the PRRSV ORF7 and Nsp9 genes revealed that the two gene mRNA expressions were decreased in cells stimulated with PoIFN-αas compared to vehicle-treated cells. The results indicated that rAd-PoIFNαcould resist PRRSV in vitro and had the potential to be used to protect swine from PRRSV in clinical.5. Porcine interferon-alpha or interferon-gamma enhances the IgG and IgG2 antibody levels and switches to the Th1 response to classical swine fever virus(CSFV) vaccine in swineTo investigate which form would significantly affect the immunostimulatory function, the utilization of IFNs were expressed in E.coli and eukaryotic expressing vector, respectively. Piglets were intramuscularly(IM) co-injected with 150 RID(rabbit infectious dosage) CSFV vaccine plus 10⁶U, 2×10⁵U and 5×10⁴U of PoIFN-αor PoIFN-γexpressed in E.coli or plus DNA plasmid encoding PoIFN-αor PoIFN-γ, respectively. After the boost immunization, the total IgG titers of groups co-immunized with PoIFN-α(2×10⁵U or 5×10⁴U) or all doses of PoIFN-γexpressed by E.coli were significantly higher than group immunized with vaccine alone. After the first immunization, groups of 10⁶U PoIFN-αand all doses of PoIFN-γexpressed by E.coli could induce statistically higher IgG2 titers than group immunized with vaccine alone. They also induced higher IgG2 titers than group co-immunized with pcD-PoIFNγ. And these groups could significantly switch the IgG2/IgG1 ratio to Th1 response after the first immunization. Moreover, only addition of 2×10⁵U PoIFN-γcould last the IgG2/IgG1 ratio to Th1 response for a longer period. The results indicated that PoIFN-γhad more powerful capability of immunostimulatory functions than PoIFN-αto induce the total IgG, IgG1 and IgG2 and to switch the response to Th1. And the IFN protein expressed in E.coli was more suitable than IFN mediated by DNA plasmid to improve the immunogenicity of CSFV vaccine as adjuvant. The effects of PoIFN-γon the titers of IgG and IgG1 were dose-dependent in negative correlation, while PoIFN-αon the titers of IgG were dose-dependent in positive correlation.