Accession Number : ADA454362


Title :   Cloning, Expression, and Purification of Brucella suis Outer Membrane Proteins


Descriptive Note : Journal article


Corporate Author : WALTER REED ARMY INST OF RESEARCH WASHINGTON DC DEPT OF BACTERIAL DISEASES


Personal Author(s) : Ding, Xuan Z ; Bhattacharjee,, Apurba ; Nikolich,, Mikeljon P ; Paulsen,, Ian T ; Myers,, Gary ; Seshadri,, Rekha ; Hoover, David L


Full Text : https://apps.dtic.mil/dtic/tr/fulltext/u2/a454362.pdf


Report Date : Jan 2005


Pagination or Media Count : 9


Abstract : Brucella, an aerobic, nonsporeforming, nonmotile Gram-negative coccobacillus, is a NIH/CDC category B bioterror threat agent that causes incapacitating human illness. Medical defense against the bioterror threat posed by Brucella would be strengthened by development of a human vaccine and improved diagnostic tests. Central to advancement of these goals is discovery of bacterial constituents that are immunogenic or antigenic for humans. Outer membrane proteins (OMPs) are particularly attractive for this purpose. In this study, we cloned, expressed, and purified seven predicted OMPs of Brucella suis. The recombinant proteins were fused with 6-His and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based on their ORF sequences and directly cloned into an entry vector. The recombinant entry constructs were propagated in TOP 10 cells, recombined into a destination vector, pET-DEST42, then transformed into E. coli BL21 cells for IPTG-induced protein expression. The expressed recombinant proteins were confirmed with Western blot analysis using anti-6-His antibody conjugated with alkaline phosphatase. These B. suis OMPs were captured and purified using a HisGrab plate. The purified recombinant proteins were examined for their binding activity with antiserum. Serum derived from a rabbit immunized intramuscularly with dialyzed cell lysate of Brucella rough mutant WRR51. The OMPs were screened using the rabbit antiserum and purified IgG. The results suggested that recombinant B. suis OMPs were successfully cloned, expressed and purified. Some of the expressed OMPs showed high binding activity with immunized rabbit antiserum.


Descriptors :   *PURIFICATION , *VACCINES , *BRUCELLA SUIS , REPRINTS , IMMUNE SERUMS , BIOLOGICAL WARFARE AGENTS , PROTEINS


Subject Categories : Genetic Engineering and Molecular Biology
      Medicine and Medical Research
      Pharmacology


Distribution Statement : APPROVED FOR PUBLIC RELEASE