| Pasteurella multocida(P.multocida)can induce a verity of animals disease which may cause heavy loss in livestock industry,therefore,to prevent and control the disease by vaccination is a vital method.Among them,P.multocida capsular serotype A and D are the predominant serotypes of swine.This study aims at researching molecular epidemiology though analyzing P.multocida candidate protein antigen,includes outer membrane protein gene(ompA,ompH,olpB,omaP),outer membrane lipoprotein gene(vacJ,plpP,plpE),filamentous hemagglutinin protein gene(fhab2),membrane transport protein gene(transmembrane protein,Tmp),TnoB-dependent receptor protein gene(Hgb,HgbA),adhesion factor(MapA)and type 4 fimbrial gene(ptfA),up to 13 kinds of protein antigen and figuring their homology and the distribution rules between serologic type A and serologic type D out,so as to preliminary judge the scope of its immune protection.Then we constructed plasmid and expressed prokaryotic for selected protective antigen gene,and immuned them with attenuated vaccine and inactivated vaccine in mice.Through intranasal and intraperitoneal injection,we can assess their immunization effects towards Pasteurella multocida.After the investigation of Molecular Epidemiology,the positive rate of omaP、Tmp、ompA、ptfA、olpB and Hgb all was 100%;while MapA、HgbA、plpE and fhab2only were detected in type A bacterial strain,which was respectively 100%、49%、40%and 42%.The positive rate of VacJ and plpP in type A bacterial strain was separately 85%and 42%,whereas their positive rate in type D bacterial strain was100%.After sequence analysis in the gene with high detection rate and plpE gene by DNAstar software,the result showed that the nucleic acid sequences homology rate of the ptfA,ompA and ompH was respectively between 97.2~100%,91.2 to 100%,and84.6 to 100%.As for Tmp,omaP,olpB,plpE and Hgb,by comparing and analyzing their homology with the existed sequence from GenBank,the results indicated that the homology of the above nucleic acid was separately 98.6~100%,96.7~100%,98.1~100%;91.6~100%;98.3~100%.Thus we know the gene homology of ptfA,ompA,Tmp,omaP,olpB and Hgb is high,up to above 90%.Above 7 kinds of protective antigenic gene were cloned into pET-28a(+)vector.The recombinant plasmid was then introduced into BL21(DE3)engineered bacteria.The results showed that there were 6 inclusion bodys which are ptfA,ompA,plpE,Tmp,omaP and Hgb,while soluble expression was 1 kand(olpB).The above proteins were purified and quantified,and the mice were immunized three times after vaccination.Accounted total weight gain of each group’s 6 mice after immunization,there was the found:attenuated vaccine(gain 12 g);immunization group ompA and Hgb(gain33 g);plpE(gain 37 g);Tmp、omaP and inactivated vaccine immunized group(weight gain 41 g);olpB(weight gain 45 g)and non-immunized group(weight gain 44g).This shows that attenuated vaccine group have influenced the growth of mice heavily than non-immunized,then engineered subunit vaccine and inactivated vaccine.Lately,after characterized the 10~7CFU porcine pasteurella multocida upon intranasal immunization of these mice,it shows that only 1 mouse in the ompA vaccine group and the plpE vaccine group died within 10 days,certifying that the immunoprotective effect of these two group were the best.About OmaP and inactivated vaccine group,3mice had died,showing that it certainly has immune protection effect.In Tmp,olpB,Hgb,ptfA,and attenuated vaccine group and non-immune challenge group,four mice were killed,showing that the immunoprotective effect was not significant.After re-selection of ompA and plpE(two recombinant protein vaccine with the best immunoprotective effect),to re-immunize mice,and characterize type A 60CFU P.multocida upon intraperitoneal inoculation,the results show that non-immunized mice were all died within 0-1.5 day after the attack,and the protection rate of immunized group plpE and ompA was 100%and 67%,respectively.In the first 1.5~4d after the attack,the protection rate of ompA immunized group decreased to 33%,then the protection rate of plpE immunization group was 100%.The protective rate of ompA was maintained at 33%after 4~10 days of challenge,while the protection rate of immunized group plpE decreased to 50%.This shows that the immune protection effect of plpE is stronger than ompA.Based on the above results,it is found that plpE and ompA are more safe and with stronger immunoprotective effect than the current commercial attenuated vaccine and other protein vaccines.Therefore selecting the above two protective antigens to perform intraperitoneal injection,we find that the protection effects of protein antigen plpE and ompA is exact which not fully protect mice against P.multocida but can reduce the incidence and delay the disease.Although there can be found that the immunoprotection effect of plpE is stronger than ompA,as per analysis of the carrier rate in the strain of the antigen,ompA gene has a wider distribution range than plpE gene and exists in the A,D capsular serotype,so ompA’s immunoprotection range is wider. |
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