Figure 2. Western blot reactivity of anti-ves1β polypeptide antibodies.

Antigen prepared from non-infected erythrocytes, IE materials from B. bovis clonal lines C9.1, CD7, CE11, and MO7, or recombinant E. coli were reacted with the antisera to identify the sizes and variant-specificity of the recognized antigens. (A) Top panel contains antigens reacted with mAb 4D9.1G1 (left half; “4D9”) and R6a-v1β765 (right half; “R6”); bottom panel contains antigens reacted with R5a-v1β765 (left half; “R5”) and pooled preimmune from rabbits R5 and R6 (right half; “Pre”). Lanes are labeled based upon the B. bovis line antigen contained, or “NRBC” for normal, uninfected red blood cell antigens. (B) R6a-v1β765 antiserum was reacted with E. coli expressing various polypeptides, either as soluble antigens or from the BugBuster-insoluble pellet. Samples contained lysate from E. coli expressing recombinant ves1β(1–765) polypeptide (rv1β765); recombinant VESA1a polypeptide (rV1a); glutathione-S-transferase (GST); or non-recombinant E. coli (Ec). The right-most lane shows a lighter exposure of the internal pellet “rv1β765” sample to facilitate visualization of major bands contained within this sample. (C) The top panel shows recognition of VESA1a in various antigenically variant B. bovis clonal lines by mAb 4D9.1G1 (4D9), whereas reactivity of the Chicken a-v1β(f1-3) with the same blot following stripping is shown in the middle panel (Cav1β). The bottom panel is a superimposition of exposures from the two probings. Gel lanes contain antigen from B. bovis IE of the CE11, CD7, and C9.1 lines, or NRBC. Note the size-polymorphism of both VESA1a and the ves1β-encoded proteins. In all panels, black arrowheads indicate VESA1b-size polypeptides, white arrowheads indicate VESA1a polypeptides, and the asterisk indicates an unexpected, size-invariant, IE-associated antigen recognized only by R5a-v1β765. Numbers to the left or right of the figures refer to molecular mass standards (in kDa).