pET3c衍生载体的构建及应用研究 |
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引用本文: | 熊盛,林剑,洪岸,刘杰森,张玲,李志英,姚汝华. pET3c衍生载体的构建及应用研究[J]. 药物生物技术, 2001, 8(1): 4-7 |
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作者姓名: | 熊盛 林剑 洪岸 刘杰森 张玲 李志英 姚汝华 |
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作者单位: | 1. 华南理工大学生物工程系,广州 510641 2. 暨南大学生物工程研究所 ,广州 510630 |
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基金项目: | 国家“九五”科技攻关重点项目(96-C02-01-03)经费资助 |
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摘 要: | 对原核表达载体pET3c进行改建,消去载体上非必要的EcoRⅠ、HindⅢ限制酶位点,然后在克隆位点BamHⅠ处引入LacZ片段,经此二步改建后的载体命名为pJN982。该载体保留了pET3c高效表达外源基因的能力,同时,其融合克隆位点由1个增至7个,并获得以目视法筛选重组子的能力。应用该载体在E.coliBL21(de3)pLysS中融合表达牛碱性成纤维细胞生长因子,表达量占菌体总蛋白30.04%。破碎菌体上清经阳离子交换和肝素亲和两步层析,得纯度为95%的重组蛋白,活性检测显示,重蛋白具有与参照品一致的促有丝分裂活性。
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关 键 词: | pET载体 多克隆位点 目视法筛选 牛碱性成纤维细胞生长因子 pET3C BBFGF |
文章编号: | 1005-8915(2001)01-004-04 |
修稿时间: | 2000-03-13 |
Constrution andApplication of A pET3c Derivative |
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Abstract: | The prokaryote expression vector pET3c was modified by destroyingthe EcoRⅠ,HindⅢ restriction enzyme sites and inserting LacZ fragment in the BamH Ⅰ site .The derivative, named as pJN982,not only retained the ability of pET3 c to express exogenous gene efficiently but also had an increased number of fus ion cloning sites from 1 to 7,as well as gained the ability to identity the rec ombinant by visual screening of E.coli colonies .Using pJN982,fused bovine basic fibroblast growth factor(B-bFGF) was expressed in E.coli BL21(DE3)pLysS to a level of 30.04% of total celluar protein . The target protein was purified by cation exchange and heparin affinity chromatography from the supernant of bacteria lysate .It was found that the recombinant B-bFGF had an identical bio-activit y with the standard bFGF. |
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Keywords: | pET vector Multiple cloning sites Visual screen BbFGF Expression |
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