| Objective To construct a double plasmid expression system of Repeats-in-toxin(RTX)domain adenylate cyclase toxin and lysine acyltransferase and to purify RTX751protein.The mouse aerosol infection model of Bordetella pertussis was used to evaluate the immunogenicity and adjuvant effect of Repeats-in-toxin(RTX)domain adenylate cyclase toxin,so as to provide some ideas for the development of a new generation of acellular pertussis vaccine.Measure The first part of the method is to obtain high purity RTX protein.adenylate cyclase toxoid(RTX751)gene fragment and CyaC were cloned from CS strain by PCR.The two genes were cloned into PET-28a and PCDFDuet-1 expression vectors respectively to construct PET-28a-RTX and PCDFDUet-1-CyaC recombinant expression plasmids,and the two plasmids were transformed into the same E.coli BL21(DE3).The recombinant RTX protein was purified by nickel column,and the RTX protein was obtained,and the antigen specificity and acetylation site were detected.The second part is to evaluate the immune protective effect of RTX.First,C57BL mice were immunized with 1/40aP+RTX and 1/80aP+RTX and 1/40aP,1/80aP,RTX and Alum,respectively,and blood samples were taken from the tail vein at an interval of 21 days and before each injection to detect the serum antibodies of IgG,IgG1 and IgG2a.Then,28 mice were continuously infected with 30min aerosol with 1X1011/CPU/mL after the second injection.Finally,the number of bacteria in lung and trachea was counted(3,7,14,28 days),and the IFN-γ,IL-17,IL-5,TNF-α cytokines and resident cells in lung and spleen tissue after infection were analyzed.Finally,the results were statistically analyzed by Grphpad prism7.0 software.Results In the first part,the two-plasmid expression system was successfully constructed,and RTX protein and CyaC protein were expressed successfully.The RTX protein with high purity was purified by nickel column and verified by Western Blot.Lys983 and Lys860 sites were not acetylated.In the second part,Serum antibody analysis showed that the specific total IgG levels of PT and FHA in 1/40aP+RTX group were significantly higher than those in 1/40aP group,the IgG2a levels of PT,FHA and PRN in 1/40aP+RTX group were significantly higher than those in 1/40aP group,the specific total IgG and IgG2a of PT,FHA and PRN in 1/80aP+RTX group were significantly higher than those in 1/80aP group,and the IgG1 of PT and PRN in 1/80aP+RTX group were significantly higher than those in 1/80aP group.The IgG2a of RTX in 1/80aP+RTX group was significantly higher than that in 1/40aP+RTX group,and the IgG2a/IgG1 of PT and FHA in l/80aP+RTX group was significantly higher than that in 1/80aP group(P<0.01).The IgG1 of RTX in);RTX group was significantly higher than that in Alum group.On the 7th day after infection,the number of bacteria in the lung of 1/40aP+RTX group was significantly lower than that of 1/40aP,1/80aP and 1/80aP+RTX groups(P<0.01),but there was no significant difference between RTX group and Alum group(P>0.05).The results of cytokine analysis showed that the addition of RTX to aP vaccine could reduce the secretion of inflammatory factors(such as IL-17,TNF-α).Conclusion This study shows that adding RTX to an appropriate dose of aP vaccine can enhance the protective effect of aP vaccine.RTX has strong immunogenicity and adjuvant effect,which can improve the antibody level of common immune antigen,shift the type of cellular response induced by aPV from Th2 to Th1/Th2 mixed type,and play an auxiliary role in bacterial clearance.Although some antibodies were produced in mice immunized with RTX alone,it had no effect on the clearance of bacteria. |
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