Establishment Of IELISA Methods To Detect Brucella Abortus OMP28Protein And Development Of The Kits

Brucellosis is a worldwide serious anthropozoonosis disease which is caused by Brucella. Outer membrane protein28(OMP28) is a sort of immunogenic Brucella protein and it can be found in different species and types of Brucella. With the development of modern biotechnology, Enzyme linked immunosorbent assay (ELISA) has became a specified method to diagnose the Brucellosis in international trade and commerce. However, the current rule in China only admit the results which are obtained from the bengal plate agglutination test, the cows whole milk ring test, the tube agglutination test or the complement fixation test. Till today, ELISA has not been assigned in our country. In this study, the protein OMP28of Brucella abortus(B. abortus) was prokaryotic expressed and purified, and the soluble recombinant protein OMP28with high purity was obtained. Then an indirect ELISA (iELISA) method for detecting antibody to B. abortus was established coating with protein OMP28, and the diagnostic kit was developed.According to the OMP28gene sequence published in GenBank, a pair of specific primers were designed. Taking the genome of B.abortus as templates, the OMP28gene was amplified by PCR. The amplified fragments were ligated to the expression vector pET-28(+) and formed the recombinant expression plasmid pET-28(+)-OMP28. After restriction enzyme identification and analysis, the recombinant expression plasmid, which was successfully constructed, transformed into recombinant expression vector E.coli BL21(DE3). Then the recombinant vector was induced to express the protein by IPTG, and the protein was identified by SDS-PAGE and soluble analysis. The results showed that the recombinant protein pET-28(+)-OMP28was soluble protein with good reactionogenicity.Then, the OMP28protein was purified by Ni-NTA spin kit and coated the ELISA plates as antigen. By optimizing the experimental conditions, an iELISA method to detect B.abortus was established. The results showed that this method had good specificity, sensitivity and repeatability. The antigen had no cross reaction with other four kinds of bovine disease serum. The standard positive samples could be detected when diluted to1:1600. The coefficient of variation within-run and between-run was low. The coincidence of the method with IDEXX reached up to100%.Next, by preservative treatment for the key laborious procedures, the iELISA kit coating with the B.abortus OMP28protein was developed. It was indicated that the kit had excellent specificity、sensitivity and repeatability. When the kits stored at4℃, the storage time was more than eight month. Clinical samples were respectively detected by the developed kits and IDEXX iELISA kits, and the coincidence rate of these two kits was96.67%. It demonstrated that the kit could be used as an alternative method for the clinical detection of B.abortus antibody.