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Microbial Immunity and Vaccines

Conformational Nature of the Borrelia burgdorferi B31 Outer Surface Protein C Protective Epitope

Robert D. Gilmore Jr., M. Lamine Mbow
Robert D. Gilmore Jr.
Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, and
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M. Lamine Mbow
Department of Pathology, Colorado State University, Fort Collins, Colorado
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DOI: 10.1128/IAI.67.10.5463-5469.1999
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    Fig. 1.

    (A) Western blot of E. coli lysates expressing recombinant OspC in the pBluescript vector. Lanes: 1, soluble sonicated fraction; 2, insoluble sonicated fraction. Approximately 10 times less protein was loaded in lane 1 than in lane 2, indicating the predominance of the recombinant OspC in the soluble fraction. Due to the small amount of OspC protein expressed by pBluescript, the antigen is observed better by immunoblotting than by Coomassie brilliant blue staining as in panel B. (B) Coomassie brilliant blue-stained SDS-PAGE gel of E. coli lysates expressing recombinant OspC in the (pET) pSCREEN-1b vector. Lanes: 1, soluble sonicated fraction; 2, insoluble sonicated fraction containing recombinant OspC inclusion bodies. Arrows indicate the recombinant OspC bands.

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    Fig. 2.

    Serological profiles of individual mice immunized with recombinant OspC expressed by the pSCREEN (pET) vector system as assayed by immunoblotting. (A) Mouse groups 1 to 5 prior to challenge; (B) mouse groups 1 to 5 at 2 weeks following challenge, with seroconversion, indicative of infection, beginning in most mice. Mice were immunized with E. coli lysate containing (pET) pSCREEN vector only with no insert (group 1), heat-denatured recombinant OspC (group 2), guanidine-HCl-denatured recombinant OspC (group 3), nondenatured, insoluble recombinant OspC (group 4), or recombinant OspC, denatured with guanidine, with removal of guanidine following antigen purification on a Ni2+cation column. Sizes of antigen bands (in kilodaltons) are given on the right. (C) Serological profile of mouse group 4, at 4 weeks following challenge, with three of five mice seroconverting, indicative of infection. Sizes of antigen bands (in kilodaltons) are given on the right.

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    Fig. 3.

    (A) Western blot demonstrating MAb B5 reactivity againstB. burgdorferi B31 low-passage-number cells (B31 low) and recombinant (Rec.) OspC proteins generated in the expression vectors pBluescript and pSCREEN. The recombinant proteins are larger than the B31 OspC due to the presence of fusion partners. Molecular masses (in kilodaltons) are given on the left. (B) Spot blot demonstrating MAb B5 reactivity against non-heat-denatured (“not boiled”) and heat-denatured (“boiled”) antigens. B31 high, B. burgdorferi B31 high-passage-number (>50 passages) cells, which produce small amounts of OspC; E. coli pSCREEN only, lysate of cells harboring the vector with no cloned insert. Recombinant (Rec.) OspC E. coli lysates are the same as in the Western blot in panel A.

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    Fig. 4.

    Diagram showing truncated constructs of theospC gene coding sequence. The long solid arrow at the top represents the coding sequence minus the signal peptide, beginning at amino acid (aa) 33. The dotted line at the left represents the missing signal peptide. The BglII restriction site is indicated. Arrows indicate the relative positions of the primers used to amplify and clone the constructs shown below. Lines bounded by dots represent the regions encoded by the truncated constructs, whose designations appear at the right. 5′ Bgl II and 3′ Bgl II were clones made by digestion of the gene with BglII. HK-12 and BS-32 were made by unidirectional deletions. 117-590, F1-590, 117-R2, and F1-R1 were made by PCR with the primers shown. (+) and (−), reactivity and no reactivity, respectively, with MAb B5.

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    Fig. 5.

    Western blot analysis of OspC expression in E. coli from truncated constructs. (A) Blot probed with the anti-OspC MAb B5. (B) The same blot stripped of antibody from panel A and reprobed with polyclonal anti-recombinant OspC derived from mice immunized with guanidine-denatured recombinant OspC. Lanes: F1-R1, pBAD construct of entire OspC coding sequence minus the leader peptide; 1, construct 117-590; 2, construct F1-590; 3, construct 117-R2; 4, pBAD vector only; 5, B. burgdorferi lysate. Arrows indicate the positions of the recombinant (rec.) OspCs (lanes 1 to 3) and theB. burgdorferi (Bb) OspC (lane 5). The recombinants are slightly larger than the native OspC due to the presence of a fusion partner from the expression vector. Construct F1-R1 expresses a doublet recombinant protein.

Tables

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  • Table 1.

    Results of infectivity assays 4 weeks postchallenge

    ImmunogenNo. of mouse samples positive for infection/ no. testeda% Protection
    E. coli with pBluescript vector only5/50
    Recombinant OspC-pBluescript
     Nondenatured1/16b93.75
     Heat denatured4/520
    Recombinant OspC-pSCREEN
     Nondenatured (insoluble inclusions)3/540
     Heat denatured5/50
     Guanidine denatured5/50
     His tag column purified (guanidine-HCl removed)5/50
    • ↵a Two assays, ear biopsy culture and Western blot, were performed, with identical results.

    • ↵b Combined results from two studies.

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Conformational Nature of the Borrelia burgdorferi B31 Outer Surface Protein C Protective Epitope
Robert D. Gilmore Jr., M. Lamine Mbow
Infection and Immunity Oct 1999, 67 (10) 5463-5469; DOI: 10.1128/IAI.67.10.5463-5469.1999

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Conformational Nature of the Borrelia burgdorferi B31 Outer Surface Protein C Protective Epitope
Robert D. Gilmore Jr., M. Lamine Mbow
Infection and Immunity Oct 1999, 67 (10) 5463-5469; DOI: 10.1128/IAI.67.10.5463-5469.1999
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KEYWORDS

Antigens, Bacterial
Bacterial Outer Membrane Proteins
Borrelia burgdorferi Group
epitopes

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