Whole Genome Sequencing Of Brucella Canis GB1 And Monoclonal Antibody Preparation And Preliminary Application

Brucellosis is an important chronic zoonotic infectious disease caused by Brucella.Brucella can be divided into 10 species,including Brucella melitensis,Brucella abortus,Brucella suis,Brucella ovis,Brucella canis and Brucella sarin in 19 biotypes,according to the difference of its antigenicity or different susceptibility to animal.Brucella canis is the first Brucella that was isolated by Carmichael from 200 samples of aborted Beagle dogs in 1966.Brucella can cause miscarriage and infection in humans,as well as monkeys and rabbits.Brucella can be divided into smooth（S-type）and rough（R-type）phenotypes.In general,Brucella melitensis,Brucella abortus,Brucella suis are smooth Brucella,while Brucella canis and Brucella sarin are naturally rough Brucella.In this study,a high-throughput sequencing technology was used to perform whole-genome sequencing of a Brucella canis strain GB1 isolated from pet dogs by our laboratory,and the gaps in GB1 strain genome were re-sequenced using relevant PCR methods.Our results indicated that the entire GB1 genome was 3,277,308bp,consisting of two chromosomes,of which one was 2,106,982bp and the other was 1,170,326bp in length,and the content of GC was 57.2%.Analysis of genome sequences demonstrated that the GB1has 3232 coding genes,67 small satellite sequences,18 microsatellite sequences and 55 tRNA.Phylogenetic analysis indicated that the GB1 strain has highest similarity to the Brucella canis strain HSK A52141 from South Korea and Brucella canis strain 2010009751 from USA.According to the COG database,2644 Brucella genes were annotated,and they were roughly divided into 22 categories.By comparing the KEGG database,1107 genes were annotated which were distributed into 92 pathways respectively.Based on the KEGG and COG annotation,most of the GB1 predictive genes were found to be related to amino acid metabolism,sugar metabolism,membrane transport and amino acid transport.Compared with Brucella canis RM6/66,49 mutations of insertions or deletion were found in GB1.We selected 4 sites for specific investigation.In order to develop a rough-type Brucella monoclonal antibody,BALB/c mice were immunized with an inactivated B.canis GB1,and hybridoma cells were obtained using a cell fusion technique.The Brucella canis GB1,Yersinia enterocolitica O:9 and E.coli O157 were ultrasonically disrupted to obtain bacterial proteins,which were subsequently used as antigen for plate coating.An ELISA assay was established to screen the expected hybridoma cells for preparation of antibody.A stable hybridoma cell line,named 4D11G7,and its ascites were prepared.The monoclonal antibody ELISA titer was 8×10⁵,and the subtype was identified as IgG_2a.Western Blot detection showed the mAb 4D11G7 not only react with the lytic antigen of rough–type GB1 and RM6/66,but also react with the smooth-type Brucella abortus 2308,Brucella melitensis 16M,Brucella suis 1330,the rough-type Brucella melitensis RM57.In addition,in this study,an assay of c-ELISA for detection of Brucella canis was initially established.The optimization of concentration of coating antigen and monoclonal antibody,dilution of enzyme-labeled antibody,sample-antigen reacting time,etc.,was studied and determined.The results showed that good specificity and sensitivity could be obtained,when the antigen was coated with a concentration of 1.0μg/well,the primary antibody was diluted to 32000-fold,and the secondary antibody was 10000×diluted.