| At present,avian influenza is widespread in China,in which avian influenza virus(AIV)of H9N2 subtype is one of the major subtypes of AIV with a wide range of transmission.Although its pathogenicity is less than that of H5 and H7 subtypes,H9N2 AIV is highly adaptable in poultry,and can be detected in almost all domestic poultry farms.Poultry infected with H9N2 AIV usually show no symptoms or mild symptoms,and are not easy to be detected.However,when the protective measures of farms are not in place,it will mix infection with other pathogenic microorganisms and cause serious economic losses.At present,the domestic H9N2 vaccines are mainly whole virus inactivated vaccines,but the immune efficacy of the vaccines needs to be improved.Therefore,it is of great clinical significance to develop an immune enhancer or adjuvant to promote the antigenicity of H9N2 AIV vaccine.Cp G dinucleotide is a kind of oligodeoxynucleotide(ODN)containing Cp G motif,which is a special site in the genome containing cytosine nucleotide followed by guanine nucleotide(-C-phosphate-G-).Oligodeoxynucleotide containing Cp G motif is called Cp G ODN,which can stimulate the immune system.Clinical studies were used to evaluate the anti-infection,anti-tumor and anti-allergic immune functions of Cp G ODN in animal models.A large number of studies have shown that Cp G ODN could be used to control various diseases of chicken,such as Salmonella,colibacillosis,avian leukemia,infectious bronchitis,Newcastle disease,avian influenza and coccidiosis.In this study,seven new Cp G ODNs with different sequences were designed using Cp G2007 as the template,and the ODN sequence with the highest stimulating activity was selected by in vitro,and then the adjuvant effect of Cp G ODNs on H9N2 AIV inactivated vaccine were detected in chickens.These experiments were designed to prove the correlation between the structural characteristics of Cp G ODN and its immune activity,and provide experimental data for improving the adjuvant strategy of AIV vaccine.This paper can be divided into the following parts.1.In vitro screening of seven newly designed Cp G ODNsFirstly,chicken embryo fibroblasts(CEF)were used as the cell model in vitro,which was treated with ODNs at the concentration of 0.25 and 1 μg/m L for 3,6 and 12 hours,respectively.The relative expression levels of the main cytokines(IL-2,IL-6,IL-12,IFN-αand IFN-γ)and the pattern recognition receptor TLR-21 of CEF were detected by fluorescence quantitative PCR(q PCR)to screen the dominant sequence of promoting cytokines in vitro.Then,two selected Cp G ODN sequences were transfected into the cells by liposome reagent,and the immune function on cytokine was verified again.q PCR results showed that when the concentration of Cp G ODN was 0.25 μg/m L,the expression levels of IL-2 and IL-12 in CEF treated with Cp G ODN1,2,4,5 and 6 were increased by more than 5 times.When the concentration of Cp G ODN was 1 μg/m L,the relative expression levels of IL-2,IL-12 and IFN-α in CEF were significantly increased by Cp G ODN5 and 6.Cp G ODN 3 could significantly increase the relative expression of IL-6 and IL-12 at 5 μg/m L.Based on these results,three optimal sequences Cp G ODN3,5 and 6 were screened.The results of liposome transfection showed that Cp G ODN3 and 5 significantly stimulated the expression of IL-6,IFN-γ and TLR-21 in CEF,and the expression of IL-6 was positively correlated with the stimulation time and concentration of Cp G ODN.In vitro experiments showed that Cp G ODN terminal with multiple guanine nucleotides(poly GS)structure have the significant effect on its inducing function,and multiple repeated TCG motifs have a prominent effect on the expression of cytokines IL-6 and IL-12.The results showed that Cp G ODN shared the strong immune effect on CEF to produce cytokines,which provided an important experimental basis for exploring the structural characteristics of Cp G ODN.2.The effect of phosphorothioate modification on the inducing function of Cp G ODNPhosphodiester backbone DNA is easily degraded by nuclease,which seriously limits the clinical application of Cp G ODN on vaccination.In order to enhance the immune effect of Cp G ODN,we modified Cp G ODN with phosphorothioate backbone(S-Cp G)and verified the inducing effect of S-Cp G in CEF cell model.CEF were treated with S-Cp G at 0.25 and1 μg/ml for 6,12 and 24 h,and the relative expression of cytokines(IL-2,IL-6,IL-12,IFN-α and IFN-γ)and TLR-21 of CEF were detected by q PCR.The results showed that S-Cp G 3and 5 had significant effects on the expression of IL-2,IL-6 and IL-12.Moreover,the expression of IL-6 was correlated with the time and concentration of S-Cp G stimulation.However,S-Cp G 6 significantly increased the expression of IFN-α and IFN-γ,in which the stimulation effect reached the maximum after 12 h.These results provided an experimental basis for the structure and function of Cp G ODN,and offered a molecular foundation for the clinical research of vaccine.3.Adjuvant effect of Cp G ODN on H9N2 avian influenza virus vaccineCp G ODN is a recent discovered adjuvant with excellent immune performance,which possess the advantages of simple structure,generally composed of more than 20 nucleotides,and easily synthesize.Additionally,various animal experiments showed that Cp G ODN could induce the humoral and cellular immunity,which were better than that of traditional adjuvants such as aluminum adjuvant.In the experiments of the first two chapters,we screened three Cp G ODN sequence with excellent immune effect.To investigate the inducing function of these Cp G ODN on vaccination,in this experiment,20-day old chicks were injected subcutaneously with S-Cp G mixed with H9N2 inactivated virus directly or S-Cp G mixed with H9N2 inactivated virus and Montanide ISA 206 VG oil adjuvant.The titer of HI antibody in serum was detected.The proliferation activity of lymphocytes was detected by MTT test.The cytokines(IL-4 and IFN-γ)and antibodies(Ig M and Ig Y)secreted by cell supernatant were detected by ELISA.The relative expression of surface active molecules(CD3,CD4,CD8,CD80,CD86,CD154 and BAAF)in spleen lymphocytes were detected by q PCR.HI results showed that the levels of HI antibodies in S-Cp G 3 and 5 experimental groups were significantly higher than that of antigen control group after three immunization without adding oil adjuvant.After two weeks of first immunization with oil adjuvant,HI antibody against AIV was produced in each group,and the levels of HI antibody in S-Cp G experimental group were significantly higher than that of antigen control group after three immunization.Furthermore,at two weeks of three immunization,the molecular levels of IL-4 and IFN-γ produced by the experimental group of AIV antigen combined with S-Cp G 3and 5 were significantly higher than those of the control group immunized with single antigen;similarly,the levels of IL-4 produced by the experimental group of AIV antigen combined with oil adjuvant combined with S-Cp G 3 and 5 were also significantly higher than those of the control group immunized with single antigen.Compared with the control group immunized with single antigen,the molecular levels of IL-4 produced by the experimental group of AIV antigen combined with S-Cp G 3 and 5 were significantly higher.The levels of Ig Y antibody in the experimental group were significantly increased.Additionally,the expressions of CD3,CD80 and CD86 were significantly increased in S-Cp G 3 combined antigen group,while the expressions of CD4 and BAAF were significantly increased in SCp G 6 combined antigen group.Also,the expressions of CD8 and CD80 in the group of AIV antigen plus oil adjuvant combined with S-Cp G 3 were significantly increased,and the expressions of CD4 and CD80 in the group of AIV antigen plus oil combined with S-Cp G 5were significantly increased,and the expressions of CD3,CD4 and CD80 in the group of AIV antigen plus oil combined with S-Cp G 6 were significantly increased.The results showed that Cp G ODN could induce the strong humoral and cellular immunity,which provides an important experimental basis for exploring the structural characteristics of Cp G ODN and the study of vaccine immune mechanism. |
PDF Full Text Request