title: Passive immunity to yersiniae mediated by anti-recombinant V antigen and protein A-V antigen fusion peptide.
abstract: LcrV (V antigen), a known unstable 37.3-kDa monomeric peptide encoded on the ca. 70-kb Lcr plasmid of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, has been implicated as a regulator of the low-calcium response, virulence factor, and protective antigen. In this study, lcrV of Y. pestis was cloned into protease-deficient Escherichia coli BL21. The resulting recombinant V antigen underwent marked degradation from the C-terminal end during purification, yielding major peptides of 36, 35, 34, and 32 to 29 kDa. Rabbit gamma globulin raised against this mixture of cleavage products provided significant protection against 10 minimum lethal doses of Y. pestis (P < 0.01) and Y. pseudotuberculosis (P < 0.02). To both stabilize V antigen and facilitate its purification, plasmid pPAV13 was constructed so as to encode a fusion of lcrV and the structural gene for protein A (i.e., all but the first 67 N-terminal amino acids of V antigen plus the signal sequence and immunoglobulin G-binding domains but not the cell wall-associated region of protein A). The resulting fusion peptide, termed PAV, could be purified to homogeneity in one step by immunoglobulin G affinity chromatography and was stable thereafter. Rabbit polyclonal gamma globulin directed against PAV provided excellent passive immunity against 10 minimum lethal doses of Y. pestis (P < 0.005) and Y. pseudotuberculosis (P < 0.005) but was ineffective against Y. enterocolitica. Protection failed after absorption with excess PAV, cloned whole V antigen, or a large (31.5-kDa) truncated derivative of the latter but was retained (P < 0.005) upon similar absorption with a smaller (19.3-kDa) truncated variant, indicating that at least one protective epitope resides internally between amino acids 168 and 275.
authors.affiliation: Department of Microbiology, Michigan State University, East Lansing 48824-1101.
authors.forename: V L
authors.initials: VL
authors.lastname: Motin
authors.forename: R
authors.initials: R
authors.lastname: Nakajima
authors.forename: G B
authors.initials: GB
authors.lastname: Smirnov
authors.forename: R R
authors.initials: RR
authors.lastname: Brubaker
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D007106:Immune Sera / Q000276:immunology; D007116:Immunization, Passive; D051379:Mice; D052899:Pore Forming Cytotoxic Proteins; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D013205:Staphylococcal Protein A / Q000276:immunology*; D015007:Yersinia / Q000276:immunology*
chemical_list: D000942:Antigens, Bacterial; D007106:Immune Sera; C043908:LcrV protein, Yersinia; D052899:Pore Forming Cytotoxic Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A