Gene Cloning, Expression And Antigenicity Identification Of Brucella Abortus And Mycobacterium Bovis Antigen

Rapid and accurate detection is the premise of the prevention, control and purification ofbovine brucellosis and tuberculosis. In order to reduce the need to eliminate the endemic areaswith bovine brucellosis and tuberculosis by means of culling, and to enhance the monitoring andprevention of bovine brucellosis and tuberculosis epidemics, and to provide kinds of safe, cheap,sensitive, and specific recombinant antigens of Brucella abortus and Mycobacterium bovisthrough the market, this study intends to clone genes of Brucella abortus and Mycobacteriumbovis antigens, construct recombinant prokaryotic expression plasmids, and practice theantigenicity identification of their recombinant antigens, thus to provide valuable antigens for thedetection of Brucella and Mycobacterium tuberculosis antibodies in bovine serum, and toestablish a foundation for the diagnosis of bovine brucellosis and tuberculosis in our country.The paper mainly includes the following three aspects of the research contents and results.Firstly, utilizing PCR technique,14antigen genes has successfully been cloned, specifically7for Brucella abortus, namely BCSP31, BP26, Cu-Zn SOD, DnaK, L7/L12, OMP17.3, P39, and7for Mycobacterium bovis, namely Ag85B, CFP10, ESAT-6, HBHA, MPB63, MPB70,MPB83.14recombinant prokaryotic expression plasmids were also developed for the14antigen genes.Gene sequencing results showed that, the14cloned target gene and the anticipated genes had ahigh homologous identity on both nucleotide sequences and amino acid sequences, which wasmore than99.3%.Secondly,14antigen recombinant prokaryotic expression plasmids were expressed in thehost strain E. coli BL21(DE3), followed by purification using Ni-NTA affinity chromatographyand electric elution method. The final recombined antigens had purity higher than90%mostlyand had a concentration of1.0~2.0mg/mL.Thirdly, through the establishment of indirect ELISA for identifying the antigenicity ofrecombinant antigens, the purified rBP26, rDnaK, rOMP17.3of Brucella abortus and rAg85BrCFP10, rESAT-6, rHBHA, rMPB70, rMPB83of Mycobacterium bovis were proved to havegood antigenicity, and to be suitable for candidate diagnosis antigens, which can providevaluable antigens for the detection of Brucella and Mycobacterium tuberculosis antibodies inbovine serum.