Production and identification of monoclonal antibodies against Brucella melitensis U-lipoprotein OMP19 / 中国人兽共患病学报

ABSTRACT

We produced and identified the monoclonal antibodies against Brucella melitensis U‐lipoprotein OMP19 .A DNA fragment coding omp19 of Brucella melitensis was amplified by PCR ,and inserted into the vector of pET‐30a(+ ) ,the result‐ant recombinant plasmid ,which we designated as pET‐30a(+ )/omp19 .We then transformed the plasmid into BL21(DE3) competent cells for the expression of the OMP19 protein .After induction with different concentrations of IPTG ,the colleted cells were analyzed by SDS‐PAGE ,and then OMP19 monoclonal antibodies were prepared through hybridoma technology . These mAbs were tested to reactivity to rOMP19 and nature membrance proteins (NMP) of Brucella melitensis by Western blot and IEST .We successfully constructed an expression vector of pET 30a(+ )/omp19 .An IPTG‐induced expression of the OMP19 protein (19 kDa in molecular weight) was demonstrated by SDS‐PAGE .The fusion protein existed in the form of solu‐ble ,and the OMP19 protein of high purity could be obtained by Ni‐NTA .Western blot assay showed that the refolded protein could be recognized by the anti‐serum against Brucella melitensis .Twenty‐three mAbs to OMP19 was produced in which 91 .30% were IgG1 ,twenty‐two (95 .65% ) mAbs could recognize nature OMP19 protein ,and eighteen (78 .26% ) mAbs could recognize NMP ,four mAbs could react with Brucella melitensis .The protein maintained good immunogenicity and twenty‐three mAbs were obtained ,which we believe provides a good protein candidate for the immunological research .