Prokaryotic Expression Of Brucella OMP16 And Establishment Of Indirect ELISA For Its Antibody

Brucellosis is a zoonotic infectious disease caused by the infection of Brucella,referred to as Brucellosis.In recent years,with the rapid development of the aquaculture industry in China and the frequent trading and circulation of animals and related animal products,the epidemic situation of Brucellosis among animals and humans have shown an upward trend.Among them,sheep Brucellosis is the most common,and sheep Brucellosis is more easily transmitted to humans.In order to ensure the sustainable and healthy development of the animal husbandry industry,the safety of animal products and the health of the people,the prevention,control and purification of brucellosis has become an inevitable trend and requirement under the current situation.The early and accurate diagnosis of Brucellosis is of great significance to the prevention and purification of Brucellosis.Therefore,the establishment of a rapid and sensitive detection method for Brucellosis has become an urgent need for the prevention and treatment of Brucellosis.Serological examination is often used for the diagnosis of a variety of diseases.Among which,the ELISA method is sensitive,simple,specifical,and it is commonly used as a fast and accurate detection method.In addition,Brucella outer membrane proteins 16(OMP16)has good immunogenicity,it is widely present in 6 species of Brucella and conserved in all biological variants.In this study,the genome of the B.suis S2 vaccine strain was used as a template,and the recombinant protein OMP16 was purified by primer design,PCR amplification,prokaryotic vector construction,IPTG induction,NTA resin chromatography column affinity chromatography,The purified OMP16 was identified and analyzed by SDS-PAGE and Western-blot,and then purified OMP16 was used as the coating antigen to optimize the screening of the best reaction conditions,and establish an indirect ELISA method for detecting Brucella antibodies.The results of the study are as follows:(1)the Brucella OMP16 recombinant protein was purified by cloning,prokaryotic expression,and induced expression,and the recombinant protein has good reactogenicity.(2)The purified recombinant protein OMP16 was used as the coated antigen to establish an i ELISA method.The best reaction conditions are: the concentration of the coated antigen was 1?g/m L,the serum dilution factor was 10 times,the dilution degree of HRPconjugated secondary antibody was 1:1000,and the reaction time of TMB was 30 min.(3)The i ELISA method for detecting Brucella goat antibodies established in this study has good sensitivity,The overall coincidence rate with the tiger red plate agglutination test reached 93%,the positive coincidence rate was 75.0%,and the negative coincidence rate was94.6%.The i ELISA method for the detection of brucellosis established in this study has good sensitivity,and this detection method has a good coincidence rate with the Tiger Red Tablet.It has the potential to be applied to the detection of brucellosis in production practice,and it can provide a detection method for the application of Brucella Omp16 gene deletion vaccine.