Screening Of Efficient Mutant Of Porcine Interferon α And Its Antiviral Effect In Vitro And In Vivo

Porcine interferon-α(PoIFN-α)has a wide range of antiviral activities,and plays an important role in the treatment of viral diseases,inhibition of tumor cell proliferation and regulation of immune function.In order to obtain the high activity of PoIFN-α,this study used the method of bioinformatics to analyze the gene sequence of PoIFN-α subtype,found the site with high change frequency of PoIFN-α allele,combined with the prediction of its tertiary structure,carried out site-specific mutation of the amino acid site of PoIFN-α.The expression and purification of PoIFN-αs were determended by E.coli Expression System.Through the determination of antiviral activity,the efficient mutant of PoIFN-α named PoIFN-α-156s.Then the antiviral effect of PoIFN-α-156s was determended in vitro and in vivo1.Construction,expression,purification and structure determination of PoIFN-α mutantIn order to obtain high activity of porcine interferon α,we downloaded the gene sequences of POIFN-α on NCBI,analyzed and compared them with bioinformatics software,and found the amino acid sites that were easy to change in the allele.Combined with the analysis and prediction of the three-level structure of porcine interferon α,we selected S38F、H40Q、F43L、N78D、C86Y、V151A and T156R.In the experiment,we designed PoIFN-α mutant.By using fusion PCR method,the natural PoIFN-α genes transformed into E.coli Rosetta to induce expression and identification The results showed that the expression form of the recombinant protein was inclusion body,and the molecular weight of the protein was about 33kDa.The secondary structure of PoIFN-α was determined by circular dichroism.The results show that the secondary structures of the two structures are all α-helix,and their secondary structures are the same.In this experiment,we successfully constructed the mutant of PoIFN-α and expressed it in E.coli2.In vitro bioassay of porcine interferon a and its mutantsThe safety concentration of PoIFN-αwas used to determine in ST cells and Vero cells by MTT method.The results showed that the effect of PoIFN-α-156s on cell proliferation was relatively small.The CC50 of PoIFN-α-156s and PoIFN-α were 505.8 μg/mL and 482.1 μg/mL respectively on Vero cells and 385.4 μg/mL and 185.5 μg/mL respectively on ST cells.The results showed that PoIFN-α-156s and PoIFN-α could inhibit the replication of VSV and PRV in Vero cells and ST cells in a dose-dependent manner.The higher the concentration of protein incubated cells,the more obvious the inhibition of virus replication,and the more obvious the inhibition of PoIFN-α-156s with PoIFN-α.There was significant difference in the ratio.The inhibitory effect of PoIFN-α-156s and PoIFN-α on VSV was more obvious in ST cells.When the survival rate of ST cells was 50%.the survival rate of Vero cells was about 20%;the inhibitory effect of PoIFN-α-156s on PRV was the same in Vero cells and ST cells,but the inhibitory effect of PoIFN-α on PRV was more obvious in Vero cells.Then we verify whether the antiviral effect is consistent with the above results from virus titer and gene copy number.PoIFN-α-156s showed a higher ability to inhibit virus replication than PoIFN-α in ST cells,and had a higher antiviral activity.3.PoIFN-α-156s inhibits VSV replication in micePoIFN-α-156s can inhibit the proliferation of VSV in vitro,whether it has the same effect in vivo.Therefore,we choose mice as our research object.We need to understand the metabolic time of porcine interferon a in mice,which will lay a foundation for further verification of its inhibition of virus replication.In the experiment,we used the pig interferon alpha quantitative detection kit to detect the serum of the mice in the experimental group.The results showed that the metabolism time of the pig interferon alpha efficient mutant poifn alpha 156s in mice was 48h,while that of the poifn alpha control group was 24h.The metabolism time of the PoIFN-α-156s group was about twice longer than that of the PoIFN-α group,indicating that the pig interferon efficient mutant we constructed was about twice longer than that of the PoIFN-α group.PoIFN-α-156s can effectively prolong its metabolism in mice.which is conducive to the better antiviral activity of PoIFN-α.In order to verify that PoIFN-α-156s can inhibit the proliferation of VSV in mice,the test group divided into three groups:negative control group,VSV infection group,VSV+PoIFN-α-156s group,VSV-PoIFN-α-156s grou.The results showed that compared with the VSV infected group,the virus content of VSV+PoIFN-α-156s group decreased significantly,showing a significant difference,indicating that PoIFN-α-156s can effectively inhibit the replication of VSV virus in mice.Through the analysis of the data,we found that the change trend of VSV in the lung,brain and spinal cord of the experimental group was basical the same,the content of VSV in the lung was higher,followed by the brain and spinal cord.Then we detected the content of VSV in the VSV-PoIFN-α-156s group.The results showed that the content of VSV in mice after inoculated with PoIFN-α-156s was significantly lower than that in the VSV infected group,which reflected that PoIFN-α-156s had a certain therapeutic effect.4.Construction,expression,purification and antiviral activity of fusion protein of PoIFN-α-156s and IgG FcIn order to obtain the fusion protein and verify whether its half-life prolonged in mice.The gene sequences of the fusion protein amplified by fusion PCR,and its antiviral activity and metabolic time measured in this study.The results showed that three fusion proteins of IFN-α and IgG Fc were successful constructed in this experiment,and two "GGGGS" flexible peptides added between them.Through the determination of antiviral activity and metabolic time of the fusion protein,confirmed that the fusion protein PoIFN-α-2GS-Fc constructed in this experiment had antiviral activity,and the metabolic time in mice prolonged about three times.