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蜘蛛拖丝蛋白基因的构建及在大肠杆菌中的表达
引用本文:李敏,章文贤,黄智华,黄建坤.蜘蛛拖丝蛋白基因的构建及在大肠杆菌中的表达[J].生物工程学报,2002,18(3):331-334.
作者姓名:李敏  章文贤  黄智华  黄建坤
作者单位:福建师范大学生物工程学院,福州,350007
基金项目:福建省重大科技项目资助 (No.2 0 0 1F0 0 6 )~~
摘    要:蜘蛛大壶腹线产生的拖丝是非常优良的纤维蛋白, 具有独特的强度和弹性。基于拖丝蛋白高度重复序列和部分cDNA序列, 合成蜘蛛拖丝蛋白基因单体, 通过头尾相连的构建策略, 得到拖丝蛋白多聚体, 与原核高效表达载体pET30a(+)连接, 转化大肠杆菌BLR(DE3), 用IPTG诱导表达。 表达产物经His.Bind树脂金属螯合亲和层析一步纯化, 纯度达90%以上, 表达量为20mg/L。SDS-PAGE和蛋白质印迹图谱显示表达产物分子量为37kD, 其值与氨基酸组分分析结果与理论推算值基本符合。 

关 键 词:蜘蛛    拖丝    基因合成    表达
文章编号:1000-3061(2002)03-0331-04
修稿时间:2001年9月1日

Study on Construct and Expression of Synthetic Genes Encoding Spider Dragline Silk in Escherichia coli
LI Min,ZHANG Wen-Xian,HUANG Zhi-Hua,HUANG Jian-Kun.Study on Construct and Expression of Synthetic Genes Encoding Spider Dragline Silk in Escherichia coli[J].Chinese Journal of Biotechnology,2002,18(3):331-334.
Authors:LI Min  ZHANG Wen-Xian  HUANG Zhi-Hua  HUANG Jian-Kun
Institution:College of Biological Engineering, Fujian Normal University, Fuzhou 350007, China. MLI2@sina.com
Abstract:Dragline spider silk produced from Nephilia clavipes major ampullate is a natural fibrous protein with specific mechanical properties such as high tensile strength and elasticity. Synthetic gene monomer encoding recombinant spider silk protein, based on the known repetitive protein sequence and partial cDNA sequence of dragline silk, was constructed and expressed. DNA monomer sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimer was cloned into pET30a(+), a prokaryotic high potency expression vector, and induced with IPTG. The protein from 8-unit repeat was produced in Escherichia coli at levels up to 20 mg/L. The protein was easily purified with high recovery by using an metal ion affinity chromatography and purity was over 90%. The results of SDS-PAGE and Western blot suggested that the mass of the expression product was about 37 kD. This value and amino acid analysis were consistent with those of theoretic calculation.
Keywords:spider  dragline silk  gene synthesis  expression
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