人层粘连蛋白α4链LG1组件融合蛋白的表达

目的表达重组人层粘连蛋白α4链LG1组件(HumanLamininAlpha4LG1Module,hLNα4LG1)蛋白并检测其抗原性。方法采用RTPCR方法从人胎盘组织扩增hLNα4LG1的cDNA片段,用TA克隆法将其插入pMD18T载体进行测序。用DNA重组技术构建原核表达载体pET28aLG1,用BL21(DE3)pET系统表达hLNα4LG1融合蛋白,SDSPAGE鉴定表达产物,用NiNTA亲和柱对表达蛋白进行纯化,并进行Westernblot分析。结果已成功获得hLNα4LG1cDNA序列,与GenBank中序列同源性为100%。12%SDSPAGE显示,经IPTG诱导后BL21(DE3)pET28aLG1总蛋白中出现一条相对分子质量为26000的新蛋白带,纯化后目的蛋白纯度达90%以上,且Westernblot可特异地检测到目的蛋白所对应转移带。结论成功表达和纯化hLNα4LG1蛋白,且具有良好的抗原性,为研究LG组件在疾病发生过程中的作用及其功能位点打下了基础。

Expression of Human Laminin Alpha4 LG1 Module Fusion Protein

Objective To express recombinant human laminin alpha4 LG1 module(hLNα4LG1) protein and determine its antigenicity.Methods Amplify the cDNA fragment of hLNα4LG1 from human placenta tissue by RT-PCR and insert into pMD-18T vector by T/A cloning.Identify the recombinant plasmid by sequencing.Construct a prokaryotic expression vector pET-28a-LG1 by DNA recombination technique and express hLNα4LG1 fusion protein in BL21(DE3)/pET system.The expressed product was identified by SDS-PAGE,purified by Ni-NTA affinity column and analyzed by Western blot.Results The cDNA fragment of hLNα4LG1 was successfully amplified.The homology of the fragment to that reported in GenBank was 100%.SDS-PAGE showed a new protein band with relative molecular weight of 26 000.After purification,the purity of the expressed protein reached above 90%,and showed good antigenicity as proved by Western blot.Conclusion hLNα4LG1 fusion protein was successful expressed and purified,and showed good antigenicity.It laid a foundation of study on the role of hLNα4LG1 in onset of diseases and the functional site of the protein.