Whole Genome Sequencing And Analysis Of Brucella Suis S2 And Study On The Diagnostic Methods Of Brucellosis

Brucellosis is a worldwide zoonotic infectious diseas,which is caused by brucella. In recent years, the zoonotic brucellosis is more and more serious in China,and results, in substantial economic losses and impacts on human public health. Thus, Brucellosis has become a high-profile public health problem.In order to control effectively this disease, the method of vaccine immunity is executed in many herds.However, these methods cannot discriminate Brucella vaccine strains from naturally acquired virulent strains. In the circumstances, a problem of control measures of the quarantine、purification and immunication in herds is found.Recently, the research showed that the method of differential diagnosis still can not effectively distinguish between Brucella suis S2 immunization and Brucella melitensis infections, so it is difficult to control brucellosis of flocks. Therefore, we analysed the Brucella suis S2, which is now the most widely-used in the market and sequenced its whole genome and carry out bioinformatics analysis. In addition, We also predicted some specific genes of the Brucella suis S2 compared with Brucella abortus and Brucella melitensis and screened out immunogenicity genes. As a consequence, we developed and verified a IELISA diagnostic kit to discriminate between Brucella suis S2 vaccinated and that had experienced natural infections with Brucella melitensis.1. In this research,we sequenced the whole genome of Brucella suis S2 by the high-throughput sequencing technologies and proceeded with bioinformatic analysis of the data. The result revealed that its genome size was 3,331,982 bp with a GC value was 57.23%, and a total of seven scaffolds and thirteen contings. The genome analysis showed that the predicted encoding genes had 3,243, mini satellite sequences had 52, microsatellite sequences had 14, tRNA has 58 and rRNA had 12. The Go classification of Brucella suis S2 genes found that the predicted S2 genes were classified as "biological process","cellular component", and"molecular function", Significantly-enriched biological process has 3,823 genes,2,585 genes were annotated as molecular functions and 1,949 genes were included in the cellular component categories.Functional classification and pathway assignment was performed by the Kyoto Encyclopedia of Genes and Genomes （KEGG）, predicted genes were assigned to 33 KEGG pathways and their expression products participated in different metabolic pathways. According to the COG database, the predicted 3,243 genes were divided into twenty categories.Examination of GO terms, KEGG pathways and COG associated with the annotated genes reveals most of them were correlated with amino acid metabolism, glycometabolism, membrane transport and amino acid transport. The annotated of Virulence Factor DataBase（VFDB） showed that there were 79 virulence-associated genes of brucella.13 resistance genes were found by the comparison of Antibiotic Resistance Genes Database（ARDB）. The effector proteins of type III secretion system（T3SS） can not predict the secreted proteins of S2.The comparative genomics research were used to compare predicted 3243 encoding genes with the whole genome of B.abortus and B. melitensis. The result showed that S2 had twenty specific genes.2. S2 had twenty specific genes and the better immunogenicity genes （GL-0002181、GL-0002189）of them were screened out. First GL-0002181, GL-0002189 and BP26 genes were amplified by the PCR and BP26 gene was defined as control group, then subcloned into the pMD19-T Vector and the correct sequences of these genes by bidirectional sequence were cloned into the prokaryotic expression vector pET-30（+） and transformed into E.coli BL21（DE3）, The recombinant expressed proteins induced by IPTG were analyzed and identified with SDS-PAGE. The results showed that there was three main recombinant protein band GL₀002181, GL₀002189 and BP26 with molecular weight 31KDa,29KDa and 32 KDa, respectively. Western-bloting analysis indicated that GL₀002181 and GL₀002189 recombinant proteins reacted with immune serum with S2 vaccine.but not reacted with naturally infected serum of Brucella melitensis.BP26 could react with immune serum as well as the serum of natural infection.3. An indirect enzyme-linked immunosorbent assay（iELISA） was established using the purified recombinant prteins GL0002181, GL₀002189 and BP26（control group） of B.abortus expressed in Escherichia coli as coating antigen, and The sensitivity and specificity of three recombinant antigens had been able to prove that GL₀002181 was 95% and 93.3%, the GL₀002189 was 95% and 95%, the BP26 was 90% and 86.7%, respectively. The indirect ELISA was used to test against 180 serum samples which were detected as positive serum with SAT and RBPT.The results indicted that the detection rate of GL₀002181, GL₀002189 and BP26 antigens were 35.5%（64/180）,33.8%（61/180） and 98.8%（176/180）, respectively. The significance analysis found that the positive detection rates had no significant differences （P>0.05） between GL₀002181 and GL₀002189 antigens and both of them would successfully apply to identified the Brucella suis vaccine strain S2.