Brucellosis Indirect ELISA Method And Establishment Of Sika Deer Mainly Deer Farming Area Of Jilin Province Brucella Serological Survey

In recent years, our deer farming industry has been rapid development, our country came to a huge income.However, China has a serious epidemic affecting the development of sika deer breeding industry.Brucellosis(Brucellosis) is one of the important infectious diseases, as common zoonoses, the disease is widely popular among our animals, not only brought great economic losses, but also the constant threat of human and social safety.This study aimed to establish a method for detecting brucellosis deer indirect enzyme-linked immunosorbent assay(iELISA).And by ELISA method established in some areas of Jilin samples collected for testing, put forward effective proposals for the region Deer brucellosis prevention.Includes the following three elements:(1) Brinell coli Bp26, Omp19, about/L12 gene cloning and sequence analysisLaboratory saved as a template Brucella strains S2,According Bp26, Omp19, L7 / L12 nucleotide sequence designed three pairs of specific primers.The sizewas obtained by PCR were 734 bp, 543 bp, 375 bp fragment, respectively connected with pMD18-T Simple Vector,After PCR, enzyme digestion and sequencing.The results show, Bp26, Omp19, L7 / L12 gene was successfully cloned into pMD18-T vector.(2) Brinell coliBp26, Omp19, L7 / L12, prokaryotic gene expression analysis and reactogenicityThe three positive recombinant plasmid clones digested connection with prokaryotic expression vector pGEX-4T-1, to build three sections of recombinant expression plasmids transformed into Escherichia coli BL21(DE3), and After IPTG induction, and explore the optimal target gene expression conditions, the use of dialysis on the target protein was purified Expression and purification of the product by SDS-PAGE analysis of the product, to obtain a molecular weight of 54 ku, 47 ku, 41 ku protein band. By Western blot analysis showed that the protein could bind with positive serum deer brucellosis incidence, Description Bp26, Omp19, L7 / L12 protein have good reactogenicity.(3) The establishment of indirect ELISA method and jilin province major breeding areas deer brucellosis serological surveyThe three segments were purified protein as coating antigen, through exploring the various parameters to optimize the reaction conditions, the initial establishment of an indirect ELISA for the detection of three antigens. By comparison with the final selection Bp26-ELISA method established in parts of the 254 samples collected for testing in Jilin Province, RBT positive rate of 8.6%, ELISA-positive detection rate was 10.2%. Opinions and propose effective prevention and treatment of brucellosis in the region.