| In recent years,the poultry industry has been expanding,and the consumption level of poultry meat and eggs is also increasing.However,the abuse of antibiotics is becoming more and more serious,various super bacteria and viruses are emerging one after another.Traditional poultry vaccines have been unable to meet the market requirements,the development of new vaccines is imminent.And the new vaccines that have been developed require adjuvants to increase their immune levels.Interferon and complement C3 d have been proven to have adjuvant effects,but the current reports are all that they are added separately.In this paper,chicken IFN-? and complement C3 d were expressed in tandem,and the antiviral activity was observed in comparison with chicken IFN-? expressed alone.In this experiment,the target genes chIFN-? and chIFN-?-C3 d were successfully obtained by PCR method,the eukaryotic recombinant plasmids pPICZ?A-chIFN-? and pPICZ?A-chIFN-?-C3 d were constructed,and the correct construction was verified by enzyme digestion and sequencing.Then the eukaryotic plasmids pPICZ?A-chIFN-?and pPICZ?A-chIFN-?-C3 d were linearized and electroporated into Pichia pastoris SDM1168.The expression of the target protein was induced by methanol,and the optimal induction time was selected.The results of SDS-PAGE showed that chIFN-?and chIFN-?-C3 d had a band at 17 KDa and 21 Kda respectively,which were consistent with the theoretical value.Western-Bloting assay also verified the correct expression of the target protein.The two recombinant proteins were purified by nickel column.The purified protein concentration of chIFN-? and chIFN-?-C3 d were 0.427 mg/mL and 1.0698 mg/mL respectively.The recombinant proteins chIFN-? and chIFN-?-C3 d expressed in the eukaryotic expression in this experiment and the recombinant proteins chIFN-? and chIFN-?-C3 d expressed in E.coli saved in the laboratory were tested in chicken embryos and chicken embryo fibroblasts on antivoral activities.Antiviral experimental in chicken embryos results showed that the four recombinant proteins all have antiviral activity,difference was extremely significant(P<0.01)compared with the virus group.At low concentrations,the effect of chIFN-?is good than chIFN-?-C3 d.At high concentrations,virus titer of chIFN-?-C3 d group decreased obviously,difference was extremely significant(P<0.01),the difference of antiviral activity of chIFN-?-C3 d in prokaryoticexpression and eukaryotic expression was not significant(P>0.05).In CEF antiviral experiment,results showed that under the same treatment,virus titers had no obvious difference between the four recombinant proteins,but the antiviral effect of chIFN-?-C3 d was better than the chIFN-?.Results of MTT assay showed that the antiviral effect of four recombinant protein were remarkable compared with the virus group,difference was extremely significant(P<0.01).The antiviral effect were increased of the four recombinant proteins with the increase of concentration,but difference was no significant(P>0.05).Overall,the antiviral effect of chIFN-?-C3 d was better than that of chIFN-?,the antiviral effect of two recombinant proteins expressed in eukaryotic expression system were better than that expressed in prokaryotic expressionsystem.In summary,we expressed the recombinant proteins chIFN-? and chIFN-?-C3 d in pichia pastoris successfuly,they all had good antiviral activity.This experiment laid the foundation for immune adjuvant function research,also provides a new train of thought for the new type of vaccine adjuvants development. |
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