Prokaryotic Expression Of Brucella Omp19Gene And Preparation Of Anti-omp19Monoclonal Antibodies

Brucellosis caused by Brucella is a zoonotic disease, causing great economic losses inanimal husbandry, and also great threat on human health. So it is currently one of the mostserious public health problem in the world. It has been proved that Brucella outer membraneproteins have antigen protective effects and immunogenicity, so it’s very important for theprevent and control of Brucellosis.Then, we chose Brucella outer membrane protein19forstudy. According to the Omp19gene sequences published in GenBank, a pair of specificprimer was designed, then the Omp19gene fragments were cloned successfully with the totalgenomic DNA as template. Then the target gene fragments were ligated into the expressionvector, and constructed the recombinant expression plasmids: pET32a-Omp19. The amplifiedOmp19gene fragments were cloned into pMD18-T simple vector, then transformed intocompetent cells E.coli DH5α. PCR amplification and ristriction enzyme digestion resultsconfirmed that the Omp19gene had been cloned into pMD18-T simple vector. Next, therecombinant expression plasmids were transformed into E.coli BL21, and after expressed, theHis-fusion protein of Brucella outer membrane Omp19was purified through nickel affinitychromatography column. Then the purified protein immunized Balb/c mice.And when thetiter reached a certain level, the spleen cells and SP2/0cells were fused in a certain proportion,and screened by ELISA to finally obtain the anti-Omp19monoclonal antibody.The results ofthis experiment will provide the necessary material for the subunit vaccine and diagnosticantigen, animal immune experiment, and the disease mechanism of cellular level.