Toru MATSUMOTO

Development of a new method to assess organophosphate insecticides exposure �`Cloning of acetylcholinesterase gene from the common carp ( Cyprinus carpio )�`

Ryo-hei YAMADA, Yoshio KERA, Shouji TAKAHASHI

We have recently reported the purification of acetylcholinesterase from common carp ( Cyprinus carpio ), and the inhibition of the purified enzyme by pesticides and flame retardants. The solubilization property and SDS-PAGE of the purified enzyme from common carp muscle indicate that the purified enzyme is a collagen - tailed asymmetric form. To develop a new in vivo assay system with carp that evaluates the effect of organophosphate on aquatic organisms, we tried to prepare the antiserum specific for carp acetylcholinesterase with the purified enzyme. However , we could not obtain the antiserum, because of the small amount of the purified enzyme. In this study, we clone the acetylcholinesterase gene of carp to obtain a large amount of the enzyme as recombinant protein.A partial cDNA fragment of the acetylcholinesterase was isolated by PCR using degenerate primers based on designed from conserved regions of ichthyic acetylcholinesterases. The 5'- and 3'- cDNA ends were obtained by rapid amplification of cDNA ends PCR ( RACE-PCR ). The isolated full-length cDNA contains a total of 2,368 bp including a 1,902-bp open reading frame that encodes a protein of 634 amino acid residues with a caluculated molecular mass of 72.0 kDa. The deduced amino acid sequence contains Exon T coded a collagen- tailed asymmetric form as same as purified emzyme from C.carpio from muscle.