Cloning,Expression And Protein Purification Of Major Outer Membrane Protein Of Brucella

Brucellosis is caused by Brucella infection and is one of the important zoonotic infectiousdiseases worldwide, belong to the second class infectious disease in China.In order to studythe relationship between the main outer membrane protein of brucella and apoptosis, thatthe cloning experiment the brucella outer membrane protein outer membrane protein Omp10,Omp16, Omp19, Omp25, Omp28, Omp31, Omp2a gene, and connected it to the carrier,PGEX4T-1and PET32a for prokaryotic expression, protein purification and relevant wasdone. Outer membrane protein of eukaryotic expression vector is constructed, and maked theouter membrane protein Omp16, Omp19, Omp28polyclonal antibody. The results are asfollows:1. By a small amount of amplification brucella, bacterial genome kit was used to extractits genome, primer design, after high-fidelity enzyme amplification, then PCR production ofbrucella major outer membrane protein gene end with A base by rTaq enzyme is obtained.Connected with pMD18T simple carrier for the night, transformation, coated board, pickcloning, bacteria, plasmid extraction, enzyme digestion identification, sent to sequencingcompany, the results showed that the cloned genes and the purpose of the corresponding genesequences in GenBank homology is as high as99%.2. For sequencing the right brucella outer membrane protein outer membrane proteinOmp10, Omp16, Omp19, Omp25, Omp28, Omp31, Omp2a genes, after enzyme digestionplastic recycling, overnight connection, transformation, coated board, pick cloning, bacteria,plasmid extraction, enzyme digestion identification, we connect its success to the prokaryoticexpression vector of pGEX4T-1and PET32a carrier. The carrier of enzyme identificationshow that the build is successful, and then transform its to prokaryotic expression strains of e.coli BL21bacteria.3. By a small amount of amplification brucella, bacterial genome kit was used to extractits genome, primer design, PCR for brucella major outer membrane protein gene, plasticrecycling purpose fragment, enzyme purpose fragment and PEGFPN-1carrier, overnight connection, conversion coated board, pick cloning, bacteria, plasmid extraction, enzymedigestion identification, sent to sequencing company, the sequencing results showed that thesuccessful getting brucella outer membrane protein in eukaryotic expression vector.4. Useing builded right prokaryotic expression vector, in different temperature, differentinduction time, different concentration under the induction of IPTG, we successfully inducedbrucella outer membrane protein Omp10, Omp16, Omp19, Omp28, Omp31, Omp2a, throughnickel affinity chromatography column, a purified HIS fusion protein of brucella outermembrane Omp16, Omp19, Omp28, through the rubber cutting brucella outer membrane andrepeated freezing and thawing method and purification of GST fusion protein Omp31.Through immune mice got the brucella outer membrane protein Omp16, Omp19, Omp28polyclonal antibody, its average antibody titer reached1:8x10~3and1:1x10~4and1:1x10~4.The results for the subunit vaccine and diagnostic antigen, animal immune experiment,and the disease mechanism of cellular level provides the necessary material.