CLONING AND DNA SEQUENCING OF A ALPHA-PHOSPHOLIPASE A_{2 INHIBITOR IN Lachesis muta muta HOMOLOGOUS TO CNF FROM THE BLOOD PLASMA OF Crotalus durissus terrificus}

Barcellos, C.J.(1), Estevão-Costa, M.I.(1), Velarde, D.T.(2), Cotta, G.A.(2), Fortes-Dias, C.L.(1)

(1)Centro de Pesquisa e Desenvolvimento e (2)Divisão de Imunobiológicos, Fundação Ezequiel Dias (FUNED), Belo Horizonte, MG.

A phospholipase A₂ inhibitor, named CNF, has been previously purified and characterized from the blood plasma of C. d. terrificus (the South American rattlesnake). In the present work we have cloned and sequenced a homologous inhibitor encoded in the liver of another Crotalidae, Lachesis muta muta (the bushmaster snake). Total RNA has been extracted from the liver of one specimen using TRIZOL (GIBCO). After RT-PCR with a commercially available kit, the cDNA was amplified by PCR using specific primers to the non-coding and carboxy-terminal regions of the CNF gene. The amplicon was cloned using a TA cloning kit and putative positive colonies were screened by PCR. Two positive clones were selected and the recombinant DNAs were purified (Wizard^âminiprep, Promega) and sequenced. The DNA and deduced protein sequences obtained were compared with available sequences of alpha-inhibitors from other snake species using the Vector NTI^âsoftware confirming the high degree of homology among them.

Financial support: FAPEMIG

CORRESPONDENCE TO:

Maria Inacia Estevão Costa, Rua São Clemente 186, Belo Horizonte, MG, CEP: 31230-460, Brasil, Email: miec@uaimail.com.br