Study On Antigenicity Of OMP10 Of Brucella Melitensis And Construction Of VirB8 Gene Deleted Strain Vector

Brucellosis is a Category B infectious diseases,which is a zoonoses caused by Brucella,mainly through the way such as mucous membranes,respiratory and digestive tract invasion of animals,animal infection after performance as male abortion,female orchitis,etc.,people are mainly infected with brucellosis through contact with diseased animals and animal products,after infection,the mainifestations are intermittent fever,perspiration,joint pain,orchitis,etc.,which will affect people's ability to work to varying degrees.To provide a theoretical basis for the development of Brucella vaccine,Brucella melitensis was isolated from the whole blood of brucellosis patients and was used as a template to bulid Brucella melitensis VirB8 gene deletion strains by use CRISPR-Cas9 and homologous recombination method to study the deletion of VirB8 gene influence on T4 SS in this study.The experiment result shows that:1.Isolation and identification the Brucella melitensis type 3 from whole blood of Brucellosis patients by use the bacteriology,phage lysis assay,AMOS-PCR and BCSP31-PCR.2.Use the Brucella melitensis as template which isolation from last step,construction the prokaryotic expression vector(Omp10-p ET28a)and eukaryotic expression vector(PCDNA3.1-Omp10),confirm that Omp10 protein possesses both antigenicity and immungenicity by use Immunofluorescence test,lymphocyte proliferation test,CCK8 test and flow cytometry.3.pBBR1 MCS-5 plasmid,Pwt Cas-9-Bacteria plasmid,artificially synthesized trc promotor and sgRNA scaffold fragment were used to construct pCas9-PBBR1-sgRNA plasmid,which could express Cas9 protein in brucella,by conventional restricti on enzyme digestion and ligation techniques.4.The homologous recombinant plasmid T19-vir B8-LR was constructed and the CRISPR-Cas9-sgRNA working plasmid and homologous recombinant plasmid were transformed into Brucella melitensis capable cells.Positive colonies were screened for PCR identification,and no VirB8 gene deletion strain was obtained.The application of CRISP-Cas9 system in Brucella melitensis gene editing needs further study.