Research On New Immunological Diagnosis Target Of Brucellosis

ObiectiveBrucellosis(or Bang’s disease) is a worldwide zoonotic disease, isa chronic infectious disease caused by brucella. Brucellosis a statutoryB infectious diseases in our country. The traditional method of laboratorytesting brucellosis, mainly depending on pathogen separation, istime-consuming, and require specific labs. So, it is inapplicable. Withthe development of science and technology, we have found that comparedwith complement fixation test(CFT) and SAT, enzyme-linked immunosorbentassay (ELISA) is more sensitive and specificity. This research use therepresentative of monoclonal proteins in Brucella as antigen to doindirect ELISA, setting up the method of ELISA with monoclonal proteinsin Brucella as antigen. This will provide foundation for researching thefast diagnostic kits of brucellosis.MethodsBased on a lot of references concerning of Brucella and infectiontarget of Brucella research by the local disease prevention and controlcenter Liboratory of Yunnan province, we choose outer membrane proteinOmp10and nucleoprotein L7/12to do the primary research.1. Cloning, expression and purification of Brucella omp10, L7/12We have design two pairs primers to amplify gene fragment, basing onthe gene of outer membrane protein omp10and nucleoprotein L7/12,whichhave published. The amplified gene fragment was double enzymes digestedand purify with BamH I and Not I, or EcoR I and Not I. The target genefragment was ligated into the expression vector PGEX-4T-1,PET32a(+),constructed recombinant expression, then transformed the expressionvector into E.coli BL21. The recombinant proteins was identified by colony PCR, double enzyme digestion method, Western-blotting, then purified andanalysed by AKTA purifier1.0chromatographic system.2.building indirect ELISA testing methodBrucella Omp10, L7/12were purified, then were used to detect humanserum and bovine serum as antigen to verify the two monoclonal proteinappliction value of the two protein, which was used to detectedbrucellosis with ELISA. We measured the protein concentration, then fixedthe best concentration of the coated antigen and secondantibody.Results: This research successfully build the prokaryotic expressionvector PET32a/Omp10,PGEX-4T-1/Omp10and PGEX-4T-1/L7/L12containingOmp10and L7/L12sequence, identifying by PCR and double-enzyme cleavage,proofing successfully insert expression vector. With IPTG inducing, E.Coli BL21has expressed the protein PGEX-4T-1/Omp10and PGEX-4T-1/L7/L12.