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CpTI蛋白在大肠杆菌中的高效融合表达及其纯化与活性测定
引用本文:杨丽琛,常团结,陈宛新,杨晓光,朱祯.CpTI蛋白在大肠杆菌中的高效融合表达及其纯化与活性测定[J].生物工程学报,2003,19(1):63-68.
作者姓名:杨丽琛  常团结  陈宛新  杨晓光  朱祯
作者单位:1. 中国疾病预防控制中心营养与食品安全所卫生部微量元素营养重点实验室,北京,100050
2. 中国科学院遗传研究所植物遗传分子操作开放实验室,北京,100101
基金项目:国家重点基础研究发展计划“973”项目 (No 2 0 0 1CB10 90 0 1及 2 0 0 1AA2 12 0 41),国家高技术研究发展计划“863”项目 (No 2 0 0 1AA2 12 0 41及 2 0 0 1AA2 12 2 91)基金资助~~
摘    要:豇豆胰蛋白酶抑制剂 (Cowpea Trypsin Inhibitor) 基因 (CpTI) 因其抗虫谱广及不易耐受等优点在植物基因工程中得到广泛应用。为进行遗传修饰食品(Genetically modified foods, GMF)中所含CpTI基因表达产物的安全性评价,我们需要在体外微生物体系中获得大量的CpTI蛋白。采用pGEX融合蛋白表达系统,将CpTI与GST的编码序列在大肠杆菌BL21中进行融合表达,表达产物达到菌体总蛋白的40%。经一步Glutathione Sephrose 4B亲和纯化,融合蛋白GSTCpTI纯度达到90%以上。将Thrombin蛋白酶与融合蛋白过夜作用,可获得切掉了GST标签的CpTI蛋白。活性测定显示GSTCpTI及CpTI蛋白均具有明显的胰蛋白酶抑制剂活性。用纯化的融合蛋白免疫家兔还可诱导产生高效价的抗体,经ELISA检测抗体滴度>1∶20000。Western Blotting 实验显示,无论是菌体超声裂解后总蛋白中的CpTI蛋白或是经纯化后的CpTI蛋白均可与自制的抗体发生特异性结合。这为进一步进行CpTI蛋白的安全性评价工作奠定了良好基础。

关 键 词:豇豆胰蛋白酶抑制剂,  遗传修饰食品,  安全性评价,  融合表达,  活性测定
文章编号:1000-3061(2003)01-0063-06
修稿时间:2002年8月5日

Fusion Expression, Purification and Bioactivity Assay of CpTI in Escherichia coli
YANG Li Chen,CHANG Tuan Jie,CHEN Wan Xin,YANG Xiao Guang,ZHU Zhen.Fusion Expression, Purification and Bioactivity Assay of CpTI in Escherichia coli[J].Chinese Journal of Biotechnology,2003,19(1):63-68.
Authors:YANG Li Chen  CHANG Tuan Jie  CHEN Wan Xin  YANG Xiao Guang  ZHU Zhen
Institution:Plant Genetic Manipulation Laboratory, Institute of Genetics, Chinese Academy of Sciences, Beijing 100101, China.
Abstract:CpTI (Cowpea Trypsin Inhibitor) is a widely used insect resistance gene in the plant genetic engineering for its high insecticidal activity and the minimal ability of the insects to evolve resistance to it. To facilitate the safety assessment of genetically modified foods (GMFs) with CpTI protein, we need to produce gram quantities of this protein in microbes. With the pGEX fusion expression system, we expressed the GST CpTI protein in E.coli BL21, which accounted for approximately 40% of germ proteins. By Glutathione Sephrose 4B affinity chromatography, GST CpTI was obtained with the purity up to 90%. Overnight incubate the fusion proteins with Thrombin protease, we got the CpTI proteins cleavage of GST tag. Both of the GST CpTI and CpTI proteins showed notable trypsin inhibitor activity. Immunization of rabbits with purified fusion protein generated high titer antibodies (>20000), measuring by ELISA. Western Blotting also showed specific Ag Ab binding band between the antiserum and the CpTI proteins no matter in the whole supersonic germ proteins or purified from the column. All these made a good ground for the further safety assessment of CpTI protein.
Keywords:CpTI  GMFs  safety evaluation  fusion expression  trypsin inhibitor activity
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