| 摘要: |
| 目的：构建FTL的原核表达载体,获得FTL纯化蛋白,制备抗体,为研究其生物学作用奠定基础。方法：采用基因重组技术将PCR扩增的FTL基因产物与原核表达载体pET30a(+)连接,转化入大肠杆菌BL21（DE3）,通过PCR、单双酶切及测序鉴定构建结果,用IPTG诱导蛋白表达,将融合蛋白纯化后,免疫日本大白兔,制备FTL多抗,采用Western印迹法检验抗体特异性。结果：成功地构建了FTL的原核表达载体,经大肠杆菌中诱导表达、镍亲和层析柱纯化,得到较纯的相对分子质量约25 800的融合蛋白,免疫日本大白兔后得到多抗血清,Western印迹结果显示此多克隆抗体与FTL蛋白特异性结合。结论：本研究获得FTL纯化蛋白,制备了FTL多克隆抗体,为进一步研究FTL的作用机制及其在肺癌组织中的表达情况奠定了基础。 |
| 关键词: 铁蛋白 原核表达 重组融合蛋白质类 色谱法，亲和 抗体 |
| DOI：10.3724/SP.J.1008.2007.01180 |
| 投稿时间：2007-03-28 |
| 基金项目:国家自然科学基金（30571552）;郑州市科技攻关项目(052SGYS33210);河南省自然科学基金(0611044900). |
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| Prokaryotic expression, purification and polyclonal antibody preparation of Ferritin light chain |
| JIN Lei,ZHANG Hui-zhen,YANG Ji-yao,ZHAO Xiang-feng,WANG Lin,WU Yi-ming* |
| () |
| Abstract: |
| Objective：To construct prokaryotic expression vector pET30aFTL, obtain purified FTL protein and to prepare antiFTL polyclonal antibody, so as to provide evidence for studying the biological function of the antibody. Methods: Gene recombination technology was used to link FTL gene (PCR product) and prokaryotic expression vector pET30a(+); the recombinant plasmid was then transformed into E.coli BL21 and the positive clones were identified by restriction enzyme digestion and PCR. Expression FTL protein was induced with IPTG and the expression product was purified. The purified FTL protein was used to immunize rabbits to obtain the antiserum. The specificity of the antibodies was examined by Western blotting. Results: The prokaryotic expression vector pET30aFTL was successfully constructed and a fusion protein, with a molecular weight of 25 800, was obtained after induction with IPTG and affinity chromatography. The antiFTL antibody was obtained from the immunized rabbits. The results of Western blotting indicated that the polyclonal antibody had high specificity to FTL protein. Conclusion: We have successfully obtained the purified FTL protein and antiFTL polyclonal antibody, which lays a foundation for further research on the biological function of FTL and expression of FTL in lung cancer tissues. |
| Key words: ferritin prokaryotic expression recombinant fusion proteins chromatography, affinity antibody |
