| Brucellosis is a zoonotic disease caused by Brucella. Brucella infection can cause some symptoms, such as fever, abortion, infertility, and dysgenesia. Brucellosis has caused huge economic losses and threatened public health in the world. It is very important to establish a rapid, accurate diagnostic methods in order to achieve early detection. It shows that there are false positives and cross reaction in current diagnosis methods for brucellosis. Because traditional diagnosis advantage of LPS as antigen and the structure of antigen is similar to other bacteria. Thus, some new technigues with high sensitivity and specificity are need to be used in clinical practice.In this study, the proteins of rHis-L7/L12, rHis-Bp26, rHis-GroEL, rHis-CuZn-SOD, rHis-P39 and rHis-Omp25 was expressed successfully by E.coli expression system, and fusion proteins were screened to evaluate their potential as stimulants in IFN-γ release assay for the detection of Brucellosis used. 1. Prokaryotic expression, purification and identification of six antigen proteins of Brucella abortusAccording to the sequence of rplL (L7/L12 gene), groEL, bp26, omp25, p39 and sodl (CuZn-SOD gene) gene of B. abortus S19, six pairs of primers were designed. The target fragments with expected sizes of 375 bp,1641 bp,753 bp,642 bp,1206 bp and 522 bp were amplified by PCR, and were cloned into pMD20-T and pCold I vector respectively. The recombinant plasmids were transformed into E.coli BL21, and the recombinant proteins were induced by IPTG and then the expression products were analyzed by SDS-PAGE. Results ofSDS-PAGE showed that the proteins were expressed successfully with a relative molecular weight about 20 kDa,65 kDa,33 kDa,28 kDa,47 kDa and 24 kDa. The expression products were purified from bacterial inclusion bodies by His*Bind affinity chromatography. Western blot analysis showed that the rHis-L7/L12, rHis-Bp26, rHis-GroEL, rHis-CuZn-SOD, rHis-P39 and rHis-Omp25 proteins could react specifically to anti-His MAb, which confirmed these protein’s immunoreactivity.2. Screening of the stimulants for IFN-y release assay in Brucellosis detection5 different recombinant proteins and Br-PPD were screened to evaluate their potential usage as stimulator in IFN-γ release assay for the detection of bovine brucellosis. Results from clinical samples tested showed that protein of L7/L12 displayed an advantaged positive correlation with ELISA results (Pearson’s r> 0.6) compared to other 5 stimulants, then following by Br-PPD which also showed descending order potential to be stimulators (Pearson’s r>0.2). According to the results of stimulating the same samples with 5 different recombinant proteins and Br-PPD in IFN-γ release assay, L7/L12 and Br-PPD showed a good total coincident rate with ELISA results because total coincident rate were up to 89.0%. What’s more, L7/L12 and Br-PPD also displayed a good sensitivity, specificity, positive coincident rate and negative coincident rate (sensitivity, specificity, positive coincident rate and negative coincident rate of L7/L12 and Br-PPD were 50.0%,96.7%,75%,90.7% and 54.8%,94.1%,70.8%,88.9%, respectively) in this assay. As for Bp26, GroEL, P39 and CuZn-SOD to stimulate the same samples, it showed a little positive coincident rate in IFN-γ release assay. Therefore, L7/L12 and Br-PPD displayed most obvious advantages as stimulants used in IFN-γ release assay for bovine brucellosis detection. |
