Genetic Evolution Analysis Of Brucella Melitensis Isolated In Zibo And The Establishment Of IELISA Detection Method

Brucellosis is caused by Brucella,which mainly leads to abortion,infertility,arthritis and other symptoms of infected animals.It is one of the most serious zoonotic infectious diseases in the world.Its detection methods have their own disadvantages.In this experiment,the whole genome of the newly isolated strain was sequenced,and the homology of the strain gene and the antigenicity of bvr S protein were predicted and analyzed by bioinformatics software.The expression vector was constructed by using this protein,and the purified protein was used as antigen to prepare indirect ELISA detection method.The purpose is to establish a high accuracy clinical primary screening detection method for Brucella.Firstly,the whole genome of Brucella Zibo local isolate was sequenced by high-throughput sequencing.The strain was identified as Brucella by multi sequence alignment analysis of 16 S RNA.The whole genome of the strain was compared with the gene sequence of the related strain,and the phylogenetic tree was constructed.It was found that the strain was Brucella melitensis.Although the strain has high homology with many strains of Brucella melitensis,the whole genome is not exactly the same as that of other known strains.Secondly,bioinformatics software was used to predict the physical and chemical properties,B-cell antigen epitope,T-cell antigen epitope and transmembrane domain of BvrS protein.A total of 23 antigenic determinants,13 B-cell linear epitopes and multiple T-cell epitopes were predicted.The results showed that BvrS protein had good antigenicity and could be used as antigen for indirect ELISA.TMNMM was used to predict the BvrS protein.It was found that the protein had transmembrane twice.The hydrophobicity of the transmembrane domain was high,which had a great impact on the protein expression.Therefore,the extracellular domain311-601 aa was selected for gene amplification and the construction of p ET-28a(+)-BvrS expression vector.The final concentration of the expressed protein was 1.78 mg/m L.The prepared protein was used as antigen to establish ieelisa detection method,and the parameters were optimized.The optimization results showed that the optimum concentration of coating protein was 1 ?g/m L,the best serum dilution is 1:25,the best secondary antibody dilution is 1:2000,and the best blocking is 5 % BSA.A total of 800 random samples from 16 cities in Shandong Province were detected by the newly established i ELISA(BvrS)method,and compared with other detection methods.The results showed that the positive coincidence rate with tiger red plate agglutination test was 83.87 %,with test tube agglutination test was80.77 %,and with c ELISA was 88.46%.The established i ELISA(BvrS)detection method has high accuracy and can be used for the preliminary clinical screening of Brucella melitensis in a large number of samples.In conclusion,the whole genome sequencing analysis showed that this strain was Brucella melitensis.Through the bioinformatics prediction analysis of its BvrS protein,it was found that this protein had good antigenicity.Using the expressed and purified BvrS protein as the coating antigen,an i ELISA(BvrS)method for brucellosis detection was constructed,which has high accuracy and can be used for the preliminary clinical screening of Brucella melitensismelitensis.