The Borrelia burgdorferi 37-kilodalton immunoblot band (P37) used in serodiagnosis of early lyme disease is the flaA gene product

Abstract

The 37-kDa protein (P37) of Borrelia burgdorferi is an antigen that elicits an early immunoglobulin M (IgM) antibody response in Lyme disease patients. The P37 gene was cloned from a B. burgdorferi genomic library by screening with antibody from a Lyme disease patient who had developed a prominent humoral response to the P37 antigen. DNA sequence analysis of this clone revealed the identity of P37 to be FlaA, an outer sheath protein of the periplasmic flagella. Recombinant P37 expression was accomplished in Escherichia coli by using a gene construct with the leader peptide deleted and fused to a 38-kDa E. coli protein. The recombinant antigen was reactive in IgM immunoblots using serum samples from patients clinically diagnosed with early Lyme disease that had been scored positive for B. burgdorferi anti-P37 reactivity. Lyme disease patient samples serologically negative for the B. burgdorferi P37 protein did not react with the recombinant. Recombinant P37 may be a useful component of a set of defined antigens for the serodiagnosis of early Lyme disease. This protein can be utilized as a marker in diagnostic immunoblots, aiding in the standardization of the present generation of IgM serologic tests.

FIG. 1

Diagrammatic representation of the B. burgdorferi P37/flaA coding sequence illustrating the positions of the primer pairs used to amplify three fragments of the gene subcloned for expression as described in the text. The numbers of amino acids (a.a.) deleted from the corresponding full-length protein coding sequence in constructs F2 and F3 are shown.

FIG. 2

SDS-PAGE and Western blot of the expressed products of the three P37 gene constructs. (A) SDS-PAGE protein profiles stained with Coomassie brilliant blue; (B) Western blot of the gel depicted in panel A probed with monospecific anti-P37. Lanes: Bb, B. burgdorferi B31 low-passage whole-cell lysate; E. coli lysates, IPTG-induced lysates containing the pSCREEN plasmid vector only and constructs F1, F2, and F3. Protein standards with molecular sizes in kilodaltons are shown to the left of panel A.

FIG. 3

IgM Western blots of serum samples against B. burgdorferi lysate (A) and against recombinant P37 expressed in an E. coli lysate (B). Lanes M contain marker antibody reflecting the position of the P37 antigen. Arrows indicate the positions of the P37 bands and the B. burgdorferi 41- and 39-kDa doublet bands. Lanes: 1 to 13, serum samples with positive reactivity to the B. burgdorferi P37 (samples in lanes 10 and 12 were reactive to recombinant P37 but the reaction was weaker); 14 to 19, serum samples from patients with early Lyme disease with no observed immunoreactivity against B. burgdorferi P37.

FIG. 4

IgM Western blots of serum samples reacted against the recombinant P37 antigen. Lanes: 1 to 9, serum samples from syphilis patients; 10 to 23, serum samples from healthy individuals. Lane M contains the marker antibody reflecting the position of the P37 antigen. An arrow indicates the recombinant P37 band.