The Screening Of Strains Erysipelothrix Rhusiopathiae Strains For Vaccine And Expression And Identification Of CbpB Protein

Swine erysipelas is a zoonotic infectious disease caused by Erysipelothrix rhusiopathiae.Vaccination is the most effective way to prevent swine erysipelas.There are26 kinds of Erysipelothrix rhusiopathiae serotypes?1a,1b,2-24 and N?in China,mainly for the 1A type?80%⁹0%?and type 2.Swine erysipelas vaccine contains inactivated vaccine?type 2?and attenuated vaccine?1 or 2?,with cross protection type 1 infection and type 2 vaccine against other serotypes.Choline binding protein B?CbpB?as Erysipelothrix rhusiopathiae surface protective antigen,plays an important role in the pathogenesis of.This study from 42 strains of Erysipelothrix rhusiopathiae isolates in Anhui?serotype was1a?,the pathogenicity,antigenicity and stability test,screening of Erysipelothrix rhusiopathiae vaccine strains;prokaryotic expression of the CbpB immunoreactive protein identification of Erysipelothrix rhusiopathiae,for the development of better vaccine immune protection and the combined vaccine,Erysipelothrix rhusiopathiae subunit vaccine to provide technical reserves.Through the mouse lethal test results,preliminary screened 4 strains of Erysipelothrix rhusiopathiae?AEr9,AEr21,AEr31 and AEr32?for the tested strains.First,the plate counting method,the modified Karber's method to determine the growth curve of strain and median lethal dose((LD₅₀).Second,preparation of inactivated whole cell using formaldehyde,preparation of rabbit immune serum by micro plate agglutination and agar diffusion test for the detection of serum agglutination titer and antibody titer;inactivated whole cell immune mice were challenged protection test,determination of immune protection rate.Third,the test strain was subcultured for 30 generations,and the tenth,twentieth,thirtieth generation strains were used to detect LD₅₀,precipitation antibody titer and immune protection rate respectively.Fourth,the above test results,screening of Erysipelothrix rhusiopathiae vaccine strains.Fifth,PCR AEr21 cbpB gene was amplified,cloned into pMD18-T vector to construct recombinant plasmid,pGEX-6P-1-cbpB,recombinant plasmid was transformed into Escherichia coli BL21,IPTG induced expression and purified by glutathione affinity chromatography,followed by SDS-PAGE and Western blot protein identification immune activity.The results showed that Erysipelothrix rhusiopathiae isolates AEr9,AEr21,AEr31 and AEr32 LD₅₀0 were 10.09 CFU/mL,35.5 CFU/mL,50.3 CFU/mL,845 CFU/mL,AEr21,AEr31 and selected preferred AEr32.The successful preparation of inactivated whole cell V-AEr21,V-AEr31 and V-AEr32,immune Rabbit anti swine erysipelas positive serum Ab-AEr21,Ab-AEr31 and Ab-AEr32,including Ab-AEr21 and Ab-AEr31 titer with Ag-AEr21,Ag-AEr31 and Ag-AEr32 were 50%⁷5%,precipitating antibody titer reached1:24,antigenicity was better than Ab-AEr32;inactivated whole cell V-AEr21 immune protection of AEr21,AEr31 was 100%⁸0%,V-AEr31 attack protection of AEr21,the AEr31 rate is 100%⁶0%,the protective immunity was superior to V-AEr32.AEr21 and AEr31 10 generations of LD₅₀0 were up to 103.39 CFU/mL and 103.64CFU/mL,and tends to be stable after thirtieth generation;inactivated whole cell V-AEr21 and V-AEr31precipitate antibody titer respectively by the tenth generation of 1:24 and 1:23 decreased to thirtieth 1:22;tenth-thirtieth generation of V-AEr21 and V-AEr31 on AEr21 attack protection the rate of 100%⁸0%and 60%respectively,and the protective rate of AEr31were 80%⁴0%and 100%⁸0%.The recombinant plasmid pGEX-6P-1-cbpB expressed in Escherichia coli BL21,obtain the size of 73 kD protein,CbpB protein molecular weight was consistent with the expected size,could react specifically with Erysipelothrix rhusiopathiae positive serum.The results showed that according to the pathogenicity,antigenicity and stability of the test results,we screened out AEr21 and AEr31 as Erysipelothrix rhusiopathiae vaccine candidate strains.The successful cloning and expression of porcine Erysipelothrix CbpB protein,the protein has immune activity.