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| 人工合成的GFM cry11B基因在大肠杆菌中表达、纯化及抗体制备 | |
| 引用本文： | 丁敏,王锋,苏军,陈志厚,陈在杰,孙明.人工合成的GFM cry11B基因在大肠杆菌中表达、纯化及抗体制备[J].农业生物技术学报,2004,12(6):714-719. |
| 作者姓名： | 丁敏 王锋 苏军 陈志厚 陈在杰 孙明 |
| 作者单位： | 1. 福建省农业科学院农业遗传工程重点实验室,福州,350003;华中农业大学农业微生物农业部重点实验室,武汉,430070 2. 福建省农业科学院农业遗传工程重点实验室,福州,350003 3. 华中农业大学农业微生物农业部重点实验室,武汉,430070 |
| 基金项目： | 国家高技术研究与发展计划(863)(No.2002AA212031)、福建省重大科技项目(No.2003N002,No.99-Z-3)和福建省自然科学基金重点项目(No.B0120003)资助. |
| 摘 要： | 摘要: 用限制性内切酶酶切的方法把人工合成的GFM cry11B基因编码区克隆到原核表达载体pGEX-4T-3中，构建成重组表达质粒pGC。在大肠杆菌(Escherichia coli )中表达了GST-GFMCry11B融合蛋白，表达量约占菌体总蛋白的21%，其表达产物主要以包涵体的形式存在。对包涵体蛋白进行可溶性处理，用Glutathione Sephrose 4B纯化出GST-Cry11B融合蛋白。Thrombin酶切后得到Cry11B蛋白，将其作为抗原免疫家兔获得了滴度（ELISA）为1∶8000的抗血清，并具有较强的特异性。 |
| 关 键 词： | 关键词: GFM cry11B基因 原核表达 融合蛋白 多克隆抗体 |
| 修稿时间： | 2003年12月23 |
Expression and Purification of GST-GFMCry11B Fusion Protein in E.Coli and Preparation of Polyclonal Antibody Against GFMCry11B |
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| DING Min,WANG Feng,SU Jun,CHEN Zhi-Hou,CHEN Zai-Jie,SUN Ming.Expression and Purification of GST-GFMCry11B Fusion Protein in E.Coli and Preparation of Polyclonal Antibody Against GFMCry11B[J].Journal of Agricultural Biotechnology,2004,12(6):714-719. | |
| Authors: | DING Min WANG Feng SU Jun CHEN Zhi-Hou CHEN Zai-Jie SUN Ming |
| Abstract: | Abstract: The coding region of GFM cry11B, fully synthesized gene, was cloned into the multi-clone sites of pGEX4T vector to produce the recombined expression plasmid pGC by the methods of restriction enzyme digestion. The recombinant plasmid was transferred into Escherichia coli BL21(DE3). GST-GFMCry11B fusion protein was obtained after adding IPTG into the growth media. SDS-PAGE analysis revealed that the fusion protein was highly expressed and accumulated up to above 21% of the total bacterial protein and the main expression production was deposited as inclusion body. The GST-GFMCry11B was purified with Glutathione Sephrose 4B after soluble treatment. GFMCry11B protein cleaved by Thrombin was collected and used as the antigen to immune the rabbits. ELISA assay showed that the titer of the prepared polyclonal antibody was 1∶8000 and had high specialties. |
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