Cloning And Expression Of The Omp31 Gene Of Brucella Ovis Strain 80/019 Isolated In Xinjiang

Primers were designed according to the putative omp31 gene sequence of an international standard strain 63/290 B.ovis, and the omp31 gene was amplified by PCR with the extracted genome of Xinjiang local strain B.ovis 80/019 as template.The amplifyied omp31 gene was ligated with PBS-T by T4 DNA ligase, and Identified by PCR and restriction endonuclesaes(REs) digestion. The obtained recombinant plasmid was named PBS-T-omp31. Sequencing revealed that the omp31 was 723bp. The nucleotide sequence showed 98.76% identity with omp31 gene of the international standard strain of B.ovis 63/290 ,99.86% identity with that of the international standard strain of B.canis and B.melitensis and 100%identity with that of the international standard strain of B.cetaceaca ,B.neotomae and B.suis. REs. Nhe I and BamH I were used to digest PBS-T-omp31 ,PET-28a and pcDNA3.1(+), and the omp31 gene of strain 80/019 was cloned into PET-28a and pcDNA3.1(+) respectively, resulted in the two expression plasmids PET-28a-omp31, pcDNA3.1-omp31. Then the recombinant PET-28a-omp31 was transformed into E.coli BL21(DE3) by heat shock, and induced with IPTG, the expressed omp31 protein of Xinjiang B.ovis (80/019) were detected by SDS-PAGE and Western-blot. The recombinant pcDNA3.1-omp31 was injected into rabbits by route of im. The Anti-Brucella antibodies was detected with Rose Bengal plate (RBP) and tube agglutination test (TAT). 11 days after the first inoculation. The TAT antibody titre reached 1 MOO. The study laid the foundation for further research on immunogenicity of omp3l and DNA vaccines of B.ovis.