摘要
The cDNA encoding a variant of α bungarotoxin was cloned from the venom glands of Bungarus multicinctus by RT PCR.The deduced protein precursor contained a 21 amino acid signal peptide and a following 74 amino acid mature protein.The signal peptide is very similar to those of short chain neurotoxins,κ neurotoxins and cardiotoxins.The amino acid sequence of the mature protein is identical to α bungarotoxin (V31),a minor variant of α bungarotoxin identified by protein sequencing technique.Furthermore,the cDNA encoding the deletion precursor of α bungarotoxin was also cloned.By use of pMAL p2,the variant was overexpressed in E coli as a soluble fusion protein and purified by sepharose 6B amylose affinity chromatography,which was confirmed by western blotting with the antisera agai nst α bungarotoxin.The recombinant variant was achieved after digestion by factor X a.It displayed about 1/6 in vivo toxicity of natural α bungarotoxin.The successful cloning and functional expression of α bungarotoxin provided a basis for the future study of structure function of long neurotoxins.
抽提银环蛇毒腺总RNA ,通过反转录PCR技术扩增出其神经毒素cDNA ,克隆并测定了全序列。银环蛇神经毒素的cDNA编码 2 1个氨基酸的信号肽和 74个氨基酸的成熟蛋白。该信号肽非常类似于短链神经毒素、κ -神经毒素和心脏毒素的信号肽。而成熟蛋白的氨基酸序列与从台湾产银环蛇中通过蛋白测序鉴定的α -bungarotoxin的氨基酸序列完全一样。此外 ,还克隆到该神经毒素缺失前体cDNA。利用麦芽糖结合蛋白融合表达系统 ,该神经毒素在大肠杆菌中得到高效的可溶性表达。表达的融合蛋白通过亲和层析得到有效纯化 ,并且通过蛋白印迹验证表达和纯化的融合蛋白。经过Xa因子酶切融合蛋白得到的重组神经毒素具有小鼠体内毒性 ,约为天然银环蛇神经毒素毒性的 1/ 6。该神经毒素的克隆和有效的功能表达为今后通过定点突变来研究长链神经毒素的结构与功能关系奠定了基础。
基金
the"95"great program of Chinese Academy of Sciences! (KY95 1-A1-3 0 1-0 2 )
