Molecular Cloning and Expression of the cDNA for Disintegrin from Agkistrodon acutus.

In order to clone novel and valuable genes from natural resources and develop genetic engineering drugs, total RNA was extracted from the venom gland of Agkistrodon acutus by one step method. The cDNAs for low molecular weight metalloproteinase were amplified by RT-PCR and cloned into the pGEMT vector. Their sequence were determined. One of the deduced amino acid sequences included a metalloproteinase and a disintegrin (acugrin) at the carboxyl terminus. This result further confirms that metalloproteinase and disintegrin are derived from the proenzyme of snake venom metalloproeinase. Acugrin exhibits a certain amino acid homology with other disintegrins from snake venoms. Acugrin has four disulfide bonds and a consensus motif RGD. The cDNA for the acugrin was cloned into the plasmid pT7ZZa and expressed in E.coli. ZZ-acugrin fusion protein could strongly inhibit aggregation of platelet induced by ADP and the IC(50) was about 4 nmol/L.