Lyme disease is the most prevalent arthropod-borne disease of humans in the United States and many other countries throughout Europe and Asia. The objectives of this project are to (1) use recombinant DNA techniques to express specific antigens of Borrelia burgdorferi to improve the serodiagnosis of Lyme disease, (2) characterize at the molecular level, isolates of the Lyme disease spirochete from a wide range of biological and geographical sources, and (3) examine adaptive molecular responses produced by B. burgdorferi during infection in ticks. Previously, we described the cloning and expression of a gene encoding a 39-kDa protein (P39) from B. burgdorferi, a causative agent of Lyme disease, which appeared to be an excellent candidate for a serological diagnostic antigen. Analysis of the completed DNA sequence revealed two adjacent open reading frames with a sequence identity of 62%. Gene 1, designated bmpA for borrelia membrane protein A, corresponds to a protein of 339 amino acids with a calculated molecular mass of 36.965 kDa and an estimated pI of 4.993. Because this gene encodes a recombinant protein that reacted by Western blot with all of 10 serum samples collected from human Lyme patients with previous erythema migrans or arthritis but not with 10 normal serum controls, it is assumed that this protein is equivalent to P39. The open reading frame of gene 2 has been designated bmpB. This open reading frame begins 116 nucleotides downstream of bmpA and potentially encodes a protein of 341 amino acids with a calculated molecular mass of 37.546 kDa, which we designate ORF2. The estimated pI is 4.859. A putative promoter 5' to the start codon of bmpA was present with E. coli consensus sigma-70 type -10 and -35 regions whereas bmpB lacked recognizable promoter elements. The deduced amino acid sequences of P39 and ORF2 were 52% identical. Both polypeptides had one or two charged amino acids after the initial methionine followed by several hydrophobic residues. At position 18 of P39 and position 15 of ORF2 there is a cysteine. This residue is preceded by the tripeptides FLS in P39 and LTS in ORF2. These sequences are consistent for a signal peptide with a signal peptidase II site at the cysteine. Both genes will be cloned separately into the pMAL expression vector, which produces a fusion product of the cloned gene and a maltose binding protein. Either fusion proteins or the purified proteins of bmpA and bmpB will then be tested for reactivity with anti-P39 antibodies. One or both of these purified recombinant proteins will then be made available for use as diagnostic antigens for Lyme disease serology tests.
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