Molecular cloning of the canine nicotinic acetylcholine receptor alpha-subunit gene and development of the ELISA method to diagnose myasthenia gravis

Vet Immunol Immunopathol. 1999 Dec 30;72(3-4):315-24. doi: 10.1016/s0165-2427(99)00140-3.


We investigated the molecular structure of canine nicotinic acetylcholine receptor (AChR) alpha-subunit gene and developed an enzyme-linked immunosorbent assay (ELISA) as an immunological method to diagnose myasthenia gravis (MG). Canine AChR alpha-subunit cDNA was constructed from mRNA isolated from skeletal muscle of five dogs using the reverse transcriptase-polymerase chain reaction and its molecular structure was determined. The canine AChR alpha-subunit gene had 1371 base pairs encoding 457 amino acids and had a 96.1% homology to the human AChR alpha-subunit gene at the amino acid level. From the results of sequencing the DNA, specific antibodies to the acetylcholine binding domain of the canine AChR alpha-subunit were produced by immunizing rabbits with synthetic oligopeptides (alpha-subunit 183-200 amino acids). The specificity of the rabbit anti-oligopeptide serum was examined by Western blot analysis using an E. coli-expressed AChR alpha-subunit protein and an AChR alpha-subunit protein fraction prepared from canine skeletal muscle as an antigen. An ELISA assay was developed using oligopeptides corresponding to the binding domain to diagnose canine MG; specific antibodies were detected from two dogs with MG, one diabetic dog and two healthy dogs among 25 dogs examined. Further examinations of the ELISA using a large number of samples of clinically MG-positive and MG-negative dogs are needed to establish its usefulness in MG diagnosis.

MeSH terms

  • Acetylcholine / metabolism
  • Amino Acid Sequence
  • Animals
  • Autoantibodies / analysis
  • Base Sequence
  • Binding Sites
  • Cloning, Molecular
  • Dogs
  • Enzyme-Linked Immunosorbent Assay / veterinary
  • Humans
  • Molecular Sequence Data
  • Muscle, Skeletal / chemistry
  • Myasthenia Gravis / genetics
  • Myasthenia Gravis / veterinary*
  • Polymerase Chain Reaction / veterinary
  • Rabbits
  • Receptors, Nicotinic / genetics*
  • Receptors, Nicotinic / immunology
  • Sequence Analysis, DNA


  • Autoantibodies
  • Receptors, Nicotinic
  • Acetylcholine

Associated data

  • GENBANK/AB021708