| 摘要: |
| [目的]进一步研究小鼠维甲酸诱导基因Ⅰ(Rig-Ⅰ)蛋白的细胞信号传导、结构与功能.[方法]利用已经构建好的逆转录病毒载体MigRI-Rig-Ⅰ,用PCR方法扩增出小鼠Rig-Ⅰ基因氨基端100 bp长度的片断(Rig-Ⅰ-N),将此片段定向克隆到原核表达载体pGEX-4T2中,构建pGEX-4T2-Rig-Ⅰ-N,再将Rig-Ⅰ基因剩余部分克隆到已构建有Rig-Ⅰ-N部分片段的pGEX-4T2-Rig-Ⅰ-N中,构建融合表达载体pGEX-4T2-Rig-Ⅰ,将其转化大肠杆菌BL21,经IPTG诱导表达,对表达产物GST-Rig-Ⅰ融合蛋白进行纯化,制备抗体,并对抗体效价进行ELISA检测及抗体特异性的Western blot分析.[结果]重组蛋白GST-Rig-Ⅰ能在大肠杆菌中表达,表达的融合蛋白GST-Rig-Ⅰ纯化和洗脱后分子质量为130 ku,纯度＞90%,制备的抗体效价达到1∶625 000,并且能够识别在原核表达系统以及真核细胞内表达的小鼠Rig-Ⅰ蛋白.[结论]建立的表达系统能够表达重组蛋白GST-Rig-Ⅰ,制备的抗体具有良好的免疫特异性. |
| 关键词: 小鼠 维甲酸诱导基因Ⅰ(Rig-Ⅰ) 原核表达 抗体制备 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金面上项目(30572110) |
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| Prokaryotic expression and purification of the Mus musculus Rig-Ⅰ protein and preparation of antibody |
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| Abstract: |
| 【Objective】 The study was done to study the signal transduction,structure and function of the protein of mouse retinoic acid induced geneⅠ(Rig-Ⅰ). 【Method】 On the basis of the constructed retrovirus plasmid MigRI-Rig-Ⅰ,the N-terminal 100 bp sequences of the mouse Rig-Ⅰ(Rig-Ⅰ-N) were amplified and cloned into the prokaryotic expression vector pGEX-4T2,formed recombinant plasmid pGEX-4T2-Rig-Ⅰ-N,the residual sequences of Rig-Ⅰwere cloned into the plasmid pGEX-4T2-Rig-Ⅰ-N to generate the recombinant expression plasmid pGEX-4T2-Rig-Ⅰ.After transforming the Escherichia coliBL21，the fusion protein was induced,purified and used to immunize rabbit,and the antibody's titer and specificity were detected with ELISA and Western blot respectively.【Result】 The fusion protein GST-Rig-Ⅰ could be expressed in the Escherichia coli,the relative molecular mass of fusion protein was 130 ku,the purity more than 90%,the titer of antibody could reach 1∶625 000 and recognize the Rig-Ⅰ protein expressed from the prokaryotic system and eukaryotic cell.【Conclusion】 Expression system pGEX-4T2Rig-Ⅰcould express recombinant protein GST-Rig-Ⅰ，antibody prepared had a good immunized specificity. |
| Key words: Mus musculus retinoic acid-induced gene Ⅰ prokaryotic expression antibody preparation |
