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中华眼镜蛇短链神经毒素cDNA的克隆及在大肠杆菌中的表达 |
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蔡 勤 何志勇 龚 毅 杨胜利
摘要:利用RT-PCR 技术从中华眼镜蛇毒腺组织中成功地克隆了短链神经毒素cDNA。测序结果表明，该基因开放阅读框架编码83个氨基酸残基，其中21个为信号肽，成熟肽为62个氨基酸残基。该基因与GenBank 报道的相同物种的神经毒素基因有相当的同源性，不同物种之间的信号肽序列十分保守。将短链神经毒素cDNA再经PCR扩增除去信号肽序列，克隆到pT7ZZ表达质粒中，转化E. coli BL21(DE3)后，经IPTG诱导可高效表达分子量为23kDa②左右的融合蛋白。表达产物占菌体总蛋白的25%左右。
关键词:神经毒素;RT-PCR;表达
中图分类号:Q75, Q812
文献标识码:A
文章编号:0253-9772(1999)05-0001-04
Cloning and Expression of a Short-chain Neurotoxin from Chinese Cobra in Escherichia coli
CAI Qin,GONG Yi,YANG Sheng-li
(1.Shanghai Research Center of Biotechnology,The Chinese Academy of Sciences,Shanghai200233,China;C
HE Zhi-yong
2.Department of Bioscience and Biotechnology,Shanghai Jiao-Tong University200240,China)
Abstract:A novel short-chain neurotoxin cDNA was cloned from Chinese cobra venom by RT-PCR. The cDNA was cloned into the pGEM-T vector and sequenced. It has a ORF encoding 83 amino acid residues and a 21 residues signal peptide. This neurotoxin gene of Chinese cobra was highly homogeneous to the short-chain neurotoxin gene of similar species reported in GenBank. Among the genes of neurotoxin from different species, the signal peptides were very conserved. The cDNA encoding the mature peptide was amplified by PCR and was cloned into pT7ZZ vector. The recombinant vector was transformed into E. coliBL2(DE3). The E. coli highly expressed the fusion protein whose mollecular weight is 23kDa, after induced by 0.1 mol/L IPTG. The expressed protein was accumulated up to more than 25% of total bacterial protein.
key words:Neurotoxin;RT-PCR;Expression
神经毒素（neurotoxin）是一类具有与神经肌肉接头的N-型乙酰胆碱受体（nAChR)结合活性的小分子蛋白质。它与nAChR结合后，阻碍化学神经递质乙酰胆碱与受体的结合，从而阻断了肌肉兴奋，导致松驰性麻痹。因此，神经毒素可以用来作为研究AChR结构、功能及其相互关系，以及研究神经传导和离子通道等极好的工具。同时神经毒素也具有较高的医用价值，如治疗重症肌无力，并且有良好的镇痛作用〔1, 2〕, 戒毒作用，抑瘤作用等〔3〕。
神经毒素根据其一级结构可以分成三类：1、短链神经毒素（60～62氨基酸残基）2, 长链神经毒素（70～74氨基酸残基）3, κ-神经毒素（66氨基酸残基）〔4〕。另一些不知其生物学活性的则称为神经毒素类似物〔5〕。到目前为止多种毒蛇的神经毒素及其类似物的c[1] [2] 下一页
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