Studies On Multilocus Sequencing Typing Of Brucella Isolates In Fujian Province And Serological Detection Of Antibodies To Brucellosis With Recombinant Antigen

Objective:Traditional biological method and MLST were used to study the typing of Brucella isolates in Fujian Province,and to explore the genetic evolution relationship of Brucella isolates in Fujian Province.The molecular epidemiological study of Brucella helps to provide scientific basis for the formulation of brucellosis prevention and control strategies.The diagnostic antigens for brucellosis were screened to establish an indirect ELISA detection method and evaluate its sensitivity and specificity,laying the foundation for the establishment of serological diagnostic methods for brucellosis.Method:1.Conventional biological identification methods were used to identify the species of Brucella isolates in Fujian Province.Genotyping of Brucella isolates in Fujian Province was carried out by MLST.Seven housekeeping genes(aroA,cobQ,dnaK,gap,glk,gyrB,trpE),1intergenic region(int-hyp)and 1 outer membrane protein gene(omp25)of Brucella were included for MLST typing.Different gene loci of Brucella isolates were amplified,and the PCR products were subjected to agarose gel electrophoresis to confirm that the specific target bands obtained and then sent for sequencing.The MLST online analysis tool were used to determine the allele type and ST type.The allelic evolutionary tree was drawn through MEGA5.0.Cluster analysis and minimum spanning tree(MST)were carried out through Bionumerics6.6 software.2.The immune relevant factors of Brucellawere selected,and the gene sequences were obtainedfrom the NCBI website.The PCR amplified target fragment was transferred into the T-A cloning vector.The truncated gene fragments with good hydrophilicity were selected and combined with the expression vector to enter the E.coli expression bacteria.After the immunoreactivity of the induced recombinant protein was identified by western blot,it was purified by electricelution.Using recombinant protein as antigen,an indirect ELISA method was established to detect antibodies of Brucella,and SAT was used as a control to evaluate its sensitivity and specificity.Result:1.Fifty Brucella isolates were distributed in various districts and cities of Fujian Province.Conventional biological identification showed that 45 strains were B.melitensis biovar 3 and5 strains were B.suis biovar 3.Of the 50 strains of Brucella isolates,44 strains were of ST8 type,5 strains were of ST17 type,and the remaining 1 strain was a new ST type: ST99.The gap gene of ST99 type was different from the existing alleles,and was defined as a new allele gap(32).The results of the MST map showed that Fujian Province shared the ST8 type with Inner Mongolia,Guangdongand Qinghai Province.2.We successfully constructed clone and expression vector of omp10,virb12,bp26 and omp31 gene of brucella and induced expression of the target protein.The western blot test proved that the full-length BP26 recombinant protein and the truncated OMP31 recombinant protein have good immunoreactivity,but the OMP31 recombinant protein cannot effectively distribute the positive and negative serum of brucellosis.The indirect ELISA test,using the BP26 recombinant protein as the antigen,was stable,specific and sensitive.Taking the SAT test as references,its detection sensitivity was 82.7% and the specificity was 95.0%,and the coincidence rate reached 91.2%.Conclusion:1.MLST typing research showed that Brucella isolates in Fujian Province was composed of ST8,ST17 and ST99 types,and the main epidemic strain was ST8 type.Among them,ST99 type was a newly discovered genotype in Fujian Province and had not been reported in other regions.2.Fujian Province shared the ST8 type with Inner Mongolia,Guangdong and Qinghai provinces,indicating that there may be circulation of sick livestock between Fujian and the above-mentioned key provinces of brucellosis.It was necessary to strengthen the relevant quarantine supervision.3.The coincidence rate of the indirect ELSIA test with BP26 recombinant protein as antigen and SAT reached 91.2%,indicating that BP26 protein had a certain potential in the diagnosis of human brucellosis and could be used as a human brucellosis diagnostic antigen.