Cloning And Prokaryocytic Expression Of Omp25 Gene Of Brucella Abortus And Measuring Of Immune Reactivity Of The Expressed Product

Brucellosis is a Brucella spp.-caused zoonosis which has occured widly in the world and has badly done harm to stock raising and human health and caused serious economic losses.. Brucella abortus is a facultative intracellular parasite organism whose primary host is cattle species, but by which human is also infected showing undulant fever. In some countries of the world, such as in China, where the disease has existed, vaccination has played a crucial role in controlling the disease. Although attenuated live vaccines against brucellosis are effective for controlling the disease, there are many potential problems such as vaccine safety for pregnant female animals and younger animals and so on. At present, it become a hotspot or focus to study and develop the new diagnostic techniques and gene engineering vaccines for controlling the disease which will show a very important significance for finally eradicating brucellosis in China.In this test, a gene recoding outer membrane protein 25 kDa (omp25) was amplified from the genomic DNA template of B.abortus by a polymerase chain reaction (PCR) technique. Sequencing of the omp25 gene proved that the nucleotide sequence of omp25 and the its speculated amino acid sequence has 100% identity with that of B.abortus reported in GeneBank, respectively, and over 98% identity with that of other species of Brucella reported in GeneBank. The PCR product was cloned into the expressing plasmid vector pGEX-6P-l. It was successfully expressed in E.coli BL21 (DE3) as a fusion protein with glutathion transferase protein at the presence of IPTG, and was identified by SDS-PAGE, and a specific protein band of 51ku was found. Western blotting and ELISA showed good immunoreactivity of the expressed product. The specific band of expression was excised from the gel and examined by the indirect hemogglutination test. The results showed positive. According to these results of the test, it was concluded that the expressed protein of omp25 gene as a fusion protein has antigenicity. From the results of predication Omp25 of Brucella have a transmambrane zone at amino acid from 6^th to 23^th; And it have a signal peptide from 14^th to 35^th amino acid of Omp25 of Brucella abortus.