Development Of The Colloidal Gold Immunochromatographic Assay Based Upon The Combined Recombinant Of Brucella Outer Membrane Proteins

Brucellosis is a zoonotic disease with high morbidity,which has a serious impact on public health.It is particularly important to detect and control the bacteria.To explore the detection technology of Brucella rapidly.Development of the colloidal gold immunochromatographic assay based upon was carried out,as follows:In this study,three recombinant plasmids pCold-TF-OMP19,pCold-TF-OMP22 and pCold-TF-OMP28 of Brucella were transformed into E.coli BL21 competent cells for expression.After purification by the exchange column technique,the purified recombinant proteins OMP19,OMP22 and OMP28 were identified by SDS-PAGE analysis,ELISA and Western blot techniques,and then the protein concentration was determined using the kit(BCA).The results showed that the sizes of the recombinant proteins OMP19,OMP22 and OMP28 were 69 KDa,74KDa and 80 KDa,respectively,which were all soluble proteins.The purity of the recombinant proteins reached more than 85% by purification of anion exchange column,and they were identified by ELISA and Western blot.The results showed that the three recombinant proteins had good reactivity and immunogenicity;The concentrations of recombinant proteins OMP19,OMP22 and OMP28 were 1.1 mg/mL,1.5 mg/mL,and 1.3 mg/mL by the BCA kit detection,respectively.Polyclonal antibody was prepared by using purified recombinant protein OMP22 immunohistochemical New Zealand white rabbit and purified using Protein A and G affinity columns,and the titer of the prepared polyclonal antibody was determined by ELISA.The results showed that the titer of polyclonal antibody detected by ELISA was1:102400.Polyclonal antibody was identified with High-purity polyclonal by SDS-PAGE electrophoresis.The concentration of polyclonal antibody detected by the kit(BCA)was 1.8 mg/mL.A colloidal gold solution with a particle size of 35 nm was prepared by citrate reduction method.The purified OMP22 and OMP19,OMP22,OMP28 mixed protein complexes were respectively used as colloidal gold marker,rabbit anti-bovine IgGand OMP22 multiantibody were prepared as the the test line and the control line.Brucella colloidal gold immunochromatographic test strips were developed by optimizing the reaction system and conditions.The results showed that the colloidal gold test strips prepared from OMP22 protein still showed clear bands when the positive serum was diluted to 32 times,and the colloidal gold test strips prepared from the labeled mixed proteins were diluted when the positive serum of Brucella bovis was diluted to 64 times,clear bands were still visible;The two test strips produced had no cross-reaction with bovine tuberculosis,bovine bluetongue,bovine viral diarrhea,cattle foot-and-mouth disease,cattle pasteurellosis,and bovine endemic leukemia positive sera;Compared with commercial kits,the coincidence rates of the test strips prepared from OMP22 protein and mixed protein and the ELISA test kit(IDEXX)were 94.2%(49/52)and 90.3%(47/52),respectively.The coincidence rate of the KERNEL antibody test card was 98%(49/50)and 94%(47/50).The test results were quick and simple.In this study,the test strips prepared were rapid and simple to use for on-site detection and screening of antibodies in Brucella.