Isolation of a cytolytic component from Naja nigricollis nigricollis [spitting cobra] venom

A cytolytic compound [Vlb] was isolated by fractionation of crude venom from Egyptian Cobra Snake, Naja nigricollis nigricollis using Sephadex G-75, followed by CM- Sephadex. The isolated protein has a cytolytic effect on red cells, stable at different temperatures [25 °C-100°C] and at different pH ranges [5-9]. It is partially pure as indicated by SDS-PAGE, with molecular weight approximately 10 kDa. It has no protease or phospholipase activity. It is a glycoprotein, with maximum absorbance at 280nm. It is non antigenic, with LD50 1.83 mg/ kg body weight. DNA extraction from mouse liver tissue and from hepatocellular carcinoma cell line [HEPG2] gave a single intact band after invitro incubation with Vlb, indicating that apoptosis is not involved in the cytotoxic mechanism. Small doses of Vlb induced lysis in breast, colon and hepatocellular carcinomas cell lines in tissue culture plates

Molecular cloning and sequencing of disintegrin like domain from Cerastes cerastes venom gland

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, Giodius 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

cDNA sequencing of fragments from fibrinolytic gene [F-4] from cerastes cerastes venom gland

A fibrinolytic anticoagulant [F-4] was previously isolated by Daoud et al; [1986] from Cerastes cerastes venom. In this study, isolation and sequencing of gene fragments of this F-4 protein was attempted. Primers utilized in the polymerase chain reactions were obtained from two sources: P1, a primer that was previously used in isolating other fibrin [ogen]olytic metalloprotease. The other primers; P2, P3 and P4 were designed using doprimer program. RT-PCR using P1 and P2 primers gave a band of 350 bp. P4 and P3 primers gave a band of 60 bp. P1 and P3 primers gave a band of 400 bp. Sequence analysis and alignment using bioinformatic programs indicated that samples 1, 2 and 3 bear significant homology to the metalloprotease family of snake venom sequences deposited in the Genbank. Translation to the amino acid sequence and alignment using protein database showed strong homology with fibrinolytic metalloproteases. Conservative domain database analysis indicated that the three sequenced samples belong to reprolysin family, which is snake venom endopeptidase requiring zinc for catalysis. Predict protein algorithm indicated one cysteine disulfide bond in sample 3. Sample 1 was described as mixed protein with 22% helix 35% extended and 41% loop structure. Sample 2 was described as all-alpha protein with 76% helix and 24% loop. Sample 3 was described as alpha-beta protein with 30% helix, 27% extended and 41% loop. The sequenced fragments of the protein were generally described as globular and seemed to be truncated of one gene related to a fibrinolytic protein