Expression Of Brucella Suis Omp31-BLS Fusion Peptide In E. Coli And Preparation Of Mouse Polyclonal Antibodies Against The Peptide

Brucellosis is an infectious zoonosis caused by Brucella, which not only invadesanimal bodies, but also contaminates food and animal goods. Brucellos is on the rise inmany area of the country, seriously impacting the meat and diary industry and humanhealth. Thus, Brucellosis is becoming a serious problem for the development of stockraising industry, even the whole economy.In this study, epitopes in Brucella membrane protein Omp31were predicted based onthe sequence analysis. By sequence alignment, we found that Brucella Omp31could bedivided into two major classes: Omp31-a and Omp31-b. The Omp31protein of Brucellasuis S2, an attenuated live vaccine used in our experiment, belonged to the Omp31-a sub-class. The protein is a transmembrane lipoprotein with a β-sheet barrel and a signal peptidesequence at the N-terminus. And two amino acid sequences48-74and193-227could serveas antigenic determinants (epitopes) for immunization.Express vectors pET-bls and pET-omp3148-74-bls were constructed for Brucellalymazine synthase protein and a fusion protein consisted of omp3148-74epitope gene fusedto bls gene. To construct the expression vectors, the27residual gene of omp3148-74and blsgene were PCR amplified with genomic DNA of Brucella suis S2as template. And thefull-length bls gene was inserted into pET-28a plasmid by double enzyme digestion andligation, resulted in expression vector pET-bls, which expressed21kD rBLS protein in E.coli upon IPTG induction as detected by SDS-PAGE. The fragment for omp31epitopegene and partial bls gene with a deletion at the N-terminus were fused together by PCR,and then the recombinant fragment was then inserted into pET-28a plasmid by doubleenzyme digestion and ligation. The resulted plasmid pET-omp3148-74-bls successfullyexpressed23kD recombinant protein rOmp3148-74-BLS in E. coli as detected by SDS-PAGE analysis.The two recombinant proteins were sued to immunize rabbits by injection. Whentested by Western blot assay, the antisera specifically recognized each of the two proteins,while the serum from the blank control rabbit did not. This result indicated that the fusionprotein rOmp3148-74-BLS can be used in Brucella vaccine in the future.