Immunogenicity Of The Recombinant Protein VP6-VP7 And Chitosan Oligosaccharide Conjugate Vaccine Of Giant Panda Rotavirus

Rotavirus(RV)can cause infectious and refractory diarrhea in human beings and lots of young animals,which leads to a high mortality rate in young animals.Currently,the precautions of rotavirus diarrhea mainly rely on the strengthening of feeding management which turns out to be unsatisfactory.Therefore,the main measure to prevent the disease lies in immunoprophylaxis.Chitosan oligosaccharides,which is characterized with good biocompatibility and solubility,plays an important role in immunomodulatory role and possesses immunopotentiating activity.Traditionally,vaccines are mainly based on simple physical mixing of antigen and adjuvant in vaccines.However,the connection of antigen and adjuvant as a whole by means of chemical coupling is conducive to strengthening the immunogenicity of vaccines to a greater extent.In this paper,chitosan oligosaccharides(COS)and recombinant protein VP6-VP7 in GPRV were coupled to evaluate the effect of the vaccine on humoral and cellular immunity through animal experiments.The main research contents of this paper are as follows:1.Construction of expression vectors like pET21b-MPB/GST/EDA-VP6-VP7,pET21b-EDA-VP6 and pET21b-EDA-VP7 by means of homologous recombinationAccording to the gene sequences of VP6 and VP7 of GPRV CH-1 strain in GenBank,homologous arms-contained primers are respectively designed.Correspondingly,the gene sequences of VP6,VP7,VP6-1 and VP7-1 are respectively obtained through RT-PCR amplification.The expression vectors after double enzyme digestion is subjected to homologous recombination reaction with corresponding fragments so as to be converted to DH5?.Then correct expression vectors like pET21b-MPB/GST/EDA-VP6-VP7,pET21b-EDA-VP6 and pET21b-EDA-VP7 are acquired after sequencing and identification.2.Induced expression,purification and protein activity analysis of Giant Panda Rotavirus recombinant proteinThe three expression vectors that have been constructed and sequenced correctly are transformed into Rosetta(DE3).Rosetta(DE3)-pET21b-EDA-VP6-VP7,pET21b-EDA-VP6,pET21b-EDA-VP7 with the best expression effect are subjected to large-scale induced expression.Then Nie-Agrose is used to purify recombinant proteins VP6-VP7,VP6 and VP7,and TEV enzyme is to remove convergency labels;in addition,the analysis on the purified recombinant proteins VP6-VP7,VP6 and VP7 by Western bolt indicate that they all possess antigenicity.3.Construction of vaccines chemically combined vaccine of Giant Panda Rotavirus recombinant protein with chitosan oligosaccharidesRecombinant proteins VP6-VP7 in GRRV are coupled with chitosan oligosaccharides with the application of click chemistry method.The coupling efficiency of the combined vaccine is verified through the measurement of analytical gel chromatography,polysaccharide protein ratio and nuclear magnetic resonance hydrogen spectrum.The results show that VP6-VP7-chitosan oligosaccharides is successfully coupled;Circular dichroism spectrum was used to identify the structural characteristics of chemical binding.The results demonstrate that there is no change for the structure of recombinant proteome in chemical binding vaccine after successful coupling.4.Immunological Characteristics of a chemical combination vaccine of Giant Panda Rotavirus recombinant protein VP6-VP7 and chitosan oligosaccharidesThe mice were immunized with GPRV VP6-VP7-chitosan oligosaccharide chemical conjugate vaccine.After the immunization experiment was completed,the serum related antibodies of the mice were determined,and the relevant cytokines were determined.The proliferation of spleen lymphocytes was detected by MTT assay.The results showed that the serum IgG antibody level in the VP6-VP7-chitosan oligosaccharide chemical binding group was significantly higher than that in the control group(p<0.05),but the stability of IgG antibody levels produced by this group is better than that of the aluminum hydroxide gel adjuvant group;The IgG antibody content produced by this group was not significantly different from that of the aluminum hydroxide gel adjuvant group(p>0.05).The level of IgA antibody in the VP6-VP7-chitosan oligosaccharide chemical binding group was significantly higher than that in other immunized groups(p<0.05).With the application of MTT method to detect the proliferation of splenic lymphocytes,the measurement results indicate that chemical binding group and adjuvant group are significantly different from other groups(p<0.05).The serum levels of IL-2 and IFN-? in the serum of immunized mice were determined.The levels of IL-2 and IFN-? in serum of VP6-VP7-chitosanoligosac charide chemical combination group were significantly higher than those in the control group(p<0.05).And the stability of IL-2 and IFN-? produced by this group is better than that of aluminum hydroxide gel adjuvant group.The serum levels of IL-4 and IL-5 in the serum of immunized mice were determined.The levels of IL-4 and IL-5 in the serum of VP6-VP7-chitosan oligosaccharide chemical combination group were significantly higher than those in the control group(p<0.05).And the stability of IL-4 produced by this group is better than that of aluminum hydroxide gel adjuvant group.VP6-VP7-chitosan oligosaccharide chemical binding vaccine has proved to be with favorable immunogenicity.Mice in the VP6-VP7-chitooligosaccharide combination group did not develop granuloma or ulcer at the injection site,Histopat hological analysis showed no pathological changes at the injection site.In conclusion,the chemical conjugate vaccine of recombinant protein VP6-VP7 modified by chitosan oligosaccharide constructed in this experiment is characterized with favorable safety,stability and immunogenicity,which provides a reference for the research of GPRV subunit vaccine.