Molecular Cloning And Sequencing Of Disintegrin Like Domain From Cerastes Cerastes Venom Gland

Abstract

Disintegrin-like domain was cloned and sequenced from Cerastes cerastes venom gland tissue. Nested RT-PCR was performed using initial primers designed based on the homology of disintegrins from Trimeresurus flavoviridis, Glodius halys , Agkistrodon halys and Trimeresurus macrosquamatus. The homology was reached using BLAST searching tool. The primers were selected using doprimers algorithm. Nested primers were those reported by Yamada et al.,(1999) for Trimeresurus flavoviridis.
The nested PCR product was approximately 150 bP as determined by agarose gel electrophoresis. Cloning of the PCR product was performed using Qiagen PCR cloning system. The construct in pDrive cloning vector was introduced into competent JM109 bacterial cells (Promega). Plasmid DNA minipreps were prepared having the 150bp insert.
Sequence analysis of the insert gave 124 bp which is in extensive sequence homology with many snake venoms disintegrin domain.
Translation of the nucleotides sequence and searching the protein database revealed amino acid sequence of disintegrin with 70% consensus identity. Prosite motif search gave two phosphorylation sites (kinase c and casein kinase 1 ) . One myristoylation site was also identified. Four cysteines seems to form disulfide bonds.


Egyptian Journal of Biochemistry and Molecular Biology Vol. 25 (2) 2007: pp. 180-191