cDNA cloning, sequence analysis,and recombinant expression of akitonin beta,a C-type lectin-like protein from Agkistrodon acutus

• 来源期刊: 中国药理学报（英文版）
• 学科: 医学
• 关键词: snake venoms cDNA akitonin C-type lectin-like proteins recombinant proteins structureactivity relationship
• 年，卷（期）: 2004,（3）
• 页码范围: 372-377
• 页数: 6页
• 分类号: R96
• 语种: 英文

摘要

AIM: To clone the cDNA of a new member of snake venom C-type lectin-like proteins, to study its structurefunction 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 pBADTOPO as vector and transformed into E. coli TOP10 competent cells. RESULTS: A novel cDNA sequence encoding akitonin β was found and accepted by GenBank (accession number AF387100). Akitonin β 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 β 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.