牦牛源大肠杆菌F17菌毛A亚基和G亚基蛋白免疫保护效果比较分析OA

In order to evaluate the immunogenicity of yak-derived Escherichia coli F17A and F17G proteins,we first used bioinformatics software,in this study,to analyze the physicochemical properties,transmembrane structure and signal peptide prediction,secondary structure and conserved structural domains prediction of the E.coli QML2206-1 F17Aand F17G proteins isolated in our laboratory.We then induced the expression of and purified the F17A and F17G proteins.Next,we administered the purified recombinant proteins to mice at high(100 μg/mouse)and low(50 μg/mouse)doses for immunization,and detected,by ELISA,the levels of specific Immunoglobulin G(IgG)antibodies and the expression levels of cytokines cluster of differentiation 4(CD4),cluster of differentiation 8(CD8),tumor necrosis factor-α(TNF-α),interferon-gamma(IFN-γ)and interleukin-4(IL-4)in mouse serum.Finally,the immunoprotective effects of the proteins were evaluated by mouse tetanus protection assay.The results showed that both proteins are stable ones with signal peptide regions.The F17A protein had no transmembrane structural domains but one fimbrial protein A(FimA)conserved structural domain,while the F17G protein had transmembrane structural domains and two conserved structural domains,FimA and Fimbrial adhesin lectin(Fim-adh-lectin).The secondary structures of the two proteins were mainly irregularly coiled.The recombinant proteins with relative molecular masses of 37 kDa and 54 kDa were successfully obtained by prokaryotic expression.After the recombinant proteins were immunized in mice,the serum-specific IgG antibody potency of the F17A high and low-dose groups reached as high as 1:32 000 and 1:16 000,respectively;and that of the F17G high- and low-dose groups reached 1:32000 and 1:25600,respectively.Vompared with the control group,the expression levels of CD4,CD8,TNF-α,IFN-γ and IL-4 in the serum of the immunized mice were elevated(P<0.05).After the mice were poisoned with the minimum lethal doses(MLD),1.5 MLD,2 MLD,and 2.5 MLD,the survival rate of the rodents in the immunized groups was 100％against the four attacking bacterial loads,except for the F17A high-dose immunized group,which only protected the mice up to a maximum of 1.5 MLD of the attacking bacterial load;and the mice in the remaining immunized groups had 100％survival against all four attacking bacterial loads.In conclusion,the F17A and F17G recombinant proteins possessed high antigenicity and some immunoprotective effects.This study provided a technical basis for further development of a genetically engineered subunit vaccine against E.coli based on the F17 protein,which would be applicable to yak.