The Coloing And Prokaryotic Expression Of HtrA And GroEL Of Xinjiang Brucella Ovis

Primers were designed according to the nucleotide sequence of HtrA and GroEL of Brucella abortus reported respectively. With the specific primers, target fragments were amplified from genomic DNA of the Xin Jiang brucella ovis via PCR.The target framents were inserted into the linearized plasmid vector pBS-T respectively. By enzyme analysis, PCR and sequencing, the recombinant plasmids were proved to be true, the recombinant plasmids of HtrA and pET- 28a were enzymed by Sac UEcoRl, in the meanwhile recombinant plasmids of GroEL and pET- 28a were digested with xhoI/BamHI,then the fragments were identified and cloned into prokaryotic expression vector pET-28a,the combinant plasmids were transformed into E.coliBL21 and induced to express with IPTG, the products of expression were detected by SDS-PAGE and western-blot.Result: the HtrA and GroEL were obtained from genomic DNA of the Xin Jiang brucella ovis. After analysis of the sequence ,the ORF of HtrA was 1542bp, the nucleotide sequence hpmology of HtrA of brucella ovis with that of Brucella abortus , Brucella melitensis, Brucella suis. was 99.68%, 99.81%, 99.55% homology respectively . The ORF of GroEL was 1642bp, and the nucleotide sequence homology of GroEL of Brucella ovis with that of Brucella abortus,Brucella melitensis,Brucella suis was 99.88%, 99.88%,99.82% respectively, the high degree of amino acid sequence homology of HtrA and GroEL were revealed.By analysis of SDS-PAGE and western-blot the fragments were cloned into prokaryotic expression vector pET-28a with the right ORF, and could express the products expected successfully in E. coli. The results of study showed: HtrA and GroEL had high homology. the products of HtrA and GroEL were abundant and could combine with serum in rabbits injected with Brucella .The results of study accorded with that of overseas. HtrA and GroEL possibily becomed candidate genes of Brucella gene engineering vaccine.