| 摘要: |
| 通过PCR扩增,从pCAMBIA1300-bar质粒中获得bar基因编码区全长,经酶切鉴定和测序后证实,bar基因分离成功。用BamH和Hind酶切,将bar基因与原核表达载体pET28a(+)连接,构建成重组质粒pET28bar。将重组质粒pET28 bar转化大肠杆菌菌株BL21(DE3)后,获得高效表达。SDS-PAGE电泳分析显示,目的蛋白主要以包涵体形式存在,蛋白质分子量约为27 ku。将包涵体离心、洗涤和纯化后,得到高纯度的目的蛋白。该蛋白可以替代植物外源蛋白进行转bar基因产品的食用安全性检测,也可用于转bar基因产品ELISA检测的抗体制备。 |
| 关键词: 抗除草剂基因bar 原核表达 蛋白纯化 转基因产品检测 |
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| 基金项目:农业部948项目“农业转基因生物及其产品安全检测技术、关键设备及相关材料的引进”(200522)；国家社会公益事业专项“转基因油菜转化特异性检测方法研究”(2005) |
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| Prokaryotic expression and purification of glufosinate resistant gene (bar) |
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| Abstract: |
| Glufosinate resistant gene (bar) has been extensively applied in the development of transgenic herbicide resistant crops.This study is designated to express the gene bar in E.coli by means of DNA recombinant technology and to produce purified foreign protein PAT for the use in food biosafety assessment and ELISA detection.The complete sequence of the gene bar was amplified by PCR from the plasmid pCAMBIA1300 bar and cloned into the recombinant expression recombinant pET28a(+) at the BamHⅠand HindⅢ.The recombinant vector pET28 bar was transformed into E.coli BL21(DE3) and expressed in high efficiency.SDS-PAGE analysis revealed that the expressed protein was in a form of inclusion bodies with the molecular weight 27 ku.After the inclusion bodies were centrifuged,washed and purified,foreign protein was obtained in high purity.This protein can be used for foodsafety assessment detection of genetically modified products with bar gene and for production of antibar antibody for ELISA detection. |
| Key words: herbicide resistant bar gene,prokaryotic expression,protein purification,GMO detection |
