The Classification Identification Of Brucella And Prokaryotic Expression Of SOD Protein And Antiserum Preparation

Brucellosis is composed of Brucella and caused by a zoonotic infectious diseases.Host range of brucella mainly includes livestock, wild animals and people, etc. The clinicalsymptoms of the disease for long-term fever, sweating, abortion and infertility, joint painand hepatosplenomegaly, etc. Brucellosis is widely popular, to the breeding companiesaround the world are with greatly economic lossing, serious threaten to human healthy atthe same time. So, to do a good job of preventing and detecting brucellosis are veryimportant.In this experiment we used PCR (polymerase chain reaction) molecular biologydetection methods of16S rDNA, omp2a gene, Cu/zinc-SOD gene, omp28, omp10geneand omp25gene, virB10application to separate and identificate the disease is expected,and also through the classification method of brucella AMOS-PCR for classification.Results showed that the53samples by polymerase chain reaction (PCR) to detect tissue,50copies of test results were negative, three positive, detection rate of5.66%, and afterparting found cattle kind of brucella in2,1kind of brucella to sheep. Through the methodof separation of bacteria can separate to2only positive, detection rate is3.77%. Theseparation of PCR detection rate than bacteria detection rate increased by1.89%, thebacteria separation method and PCR method between the coincidence rate was100%.To analyze the immunogenicity of brucella related protein, our experiments usedB.abortus vaccine strain104M as a template, by polymerase chain reaction (PCR)amplification omp28and Cu/zinc-SOD genes, and cloned them to pGEX-4t-1(+) andpET32a vector for protein expression and purification of restructuring.After series of activate detection we found that restructuring pGEX-SOD protein hada good reactive with brucella positive serum from cattle.With restructe pGEX-SOD proteinas envelope antigen and by indirect ELISA to detect it can distinguish between negativeand positive serum of brucella. Using restructure pGEX-SOD protein immune BABL/Cmice preparation for polyclonal antibody, preparation of purified polyclonal antibody. Toconstruct eukaryotic expression vector PCI-neo-SOD, extract ultra pure plasmidtransfection293t cells, IFA test showed a certain reactive with cells after transfection.All experiments showed that Cu/zinc-SOD gene of brucella can be used as thedetection for brucellosis, and the restruction of brucella Cu/zinc-SOD protein has a good immunogenicity. Suggest that using brucella Cu/zinc-SOD gene and its encoding protein toestablish brucella etiology detection diagnosis methods have very important significance.