RESUMEN
AIM: To clone cDNAs of thrombin-like enzymes (TLEs) from venom gland of Deinagkistrodon acutus and analyze the mechanisms by which their structural diversity arose. METHODS: Reverse transcription-polymerase chain reaction and gene cloning techniques were used, and the cloned sequences were analyzed by using bioinformatics tools. RESULTS: Novel cDNAs of snake venom TLEs were cloned. The possibilities of post-transcriptional recombination and horizontal gene transfer are discussed. A phylogenetic tree was constructed. CONCLUSION: The cDNAs of snake venom TLEs exhibit great diversification. There are several types of structural variations. These variations may be attributable to certain mechanisms including recombination.
Asunto(s)
ADN Complementario/genética , Trombina/genética , Venenos de Víboras/química , Viperidae , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Homología de Secuencia , Trombina/aislamiento & purificación , Viperidae/genéticaRESUMEN
AIM: To clone the cDNA of a new member of snake venom C-type lectin-like proteins, to study its structure-function relationships and to achieve its recombinant production. METHODS: PCR primers were designed based on the homology and cDNA was amplified by RT-PCR using total RNA from snake venom gland as the template. The PCR products were cloned into the plasmid pGEM-T and sequenced. The deduced protein sequence was analyzed with some bioinformatic programs. A recombinant expression plasmid was constructed using pBAD-TOPO as vector and transformed into E.coli TOP10 competent cells. RESULTS: A novel cDNA sequence encoding akitonin beta was found and accepted by GenBank (accession number AF387100). Akitonin beta consists of a typical carbohydrate recognition domain (CRD) of C-type lectins, and it is homologous with other snake venom C-type lectin-like proteins. It was predicted to be a platelet antagonist. Upon induction with arabinose rAkitonin beta expressing in E coli was achieved at a high level (superior to 150 mg/L). The recombinant fusion protein exhibited inhibitory activities on rat platelet aggregation in vitro. CONCLUSION: A new member of snake venom C-type lectin-like proteins was discovered and characterized, and an efficient recombinant expression system was established for its production.