The Separation Of Brucella,Cloning And Prokaryotic Expression Of BP26Gene In Yanbian

Brucellosis is a zoonoses which caused by Brucella. It mainly occurs in livestock such as cattle, sheep, pig etc. and human. The main clinical symptom is the inflammation of the reproductive organs and fetal membranes causing miscarriage, infertility and local lesions of various tissues. The disease is widely distributed around the world. The world economic losses caused by brucellosis is up to over30billion dollars a year. Therefore, the disease is subject to a worldwide public health problem. In recent years, the endemic areas of Brucellosis in Yanbian is gradually expanded. The morbidity and infection rates is rising. It has become one of the important epidemic disease threat to the development of cattle industry in Yanbian. It is necessary to establish the separation, detection and prevention methods of local Brucella.A Brucella strain was isolated from bovine afterbirth in Brucella selective medium. In order to indentify the bacteria, biochemical identification (H2O2test, urease oxidation test and urease test), Giemsa staining, Kozlowski staining and AMOS-PCR diagnostic kit were applied. The results of all the above test showed that the isolated bacteria is Brucella.According to Brucella bp26gene published in GenBank, a pair of specific primers were designed and synthesized. bp26gene was amplified by PCR using total DNA of Brucella. The gene was was cloned into the pMD18-T vector and subcloned into pGEX-4T-1prokaryotic expression vector to construct pGEX-4T-bp26. The correct recombinant expression vector was transformed into E. coli BL21. The expressed products were analyzed by SDS-PAGE and Western blot. SDS-PAGE showed that the size of expressed protein is approximately53KDa. Western blotting analysis indicated that the protein had good reactogenicity. This research provides a theoretical and experimental basis for the further study.