【摘要】 目的　利用基因工程技术获得重组人层粘连蛋白α4链LG3 4组件(humanlamininAlpha4LG3 4Module ,hLNα4LG3 4)蛋白并检测其抗原性。方法　采用RT PCR方法从人胎盘组织扩增hLNα4LG3 4的cDNA片段,T A克隆法将其插入pMD 18T载体进行测序。亚克隆法构建原核表达载体pET 2 8a LG3 4,在BL2 1(DE3 )中表达hLNα4LG3 4融合蛋白。融合蛋白经SDS PAGE鉴定、Ni NTA亲和层析纯化及Western印迹分析。结果　成功获得hLNα4LG3 4cDNA片段,与Gen Bank中序列同源性为98% ,突变碱基不改变蛋白的氨基酸序列。12 %SDS PAGE电泳检测显示:经IPTG诱导后BL2 1(DE3 ) /pET2 8a LG3 4细菌裂解液总蛋白中出现一条分子量为44×10 3的新蛋白带,纯化后目的蛋白纯度达95 %以上,且Western印迹可特异地检测到目的蛋白所对应转移带。结论　成功表达和纯化了hLNα4LG3 4蛋白,从而为研究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。更多还原

【Abstract】 Objective To express and detect the antigenicity of human laminin alpha4 LG3-4 module (hLNα4LG3-4) protein by gene engineering techniques. Methods The cDNA encoding hLNα4LG3-4 was amplified by RT-PCR from human placenta, then inserted into pMD-18T vector by T/A cloning and sequenced. Prokaryotic expression vector pET-28a-LG3-4 was constructed by recombinant DNA technique. The hLNα4LG3-4 fusion protein expressed in BL21(DE3)/pET system was identified by SDS-PAGE, purified by Ni-NTA resin, and assayed by Western blotting. Results The cDNA fragment of hLNα4LG3-4 was cloned successfully. While BL21 (DE3)/pET28a-LG3-4 bacterium was induced with IPTG, a new protein band with a relative molecular weight of 44 000 was shown on SDS-PAGE profile. hLNα4LG3-4 fusion protein of high purity (95%) was obtained and specific protein band was detected by Western blotting. Conclusion The hLNα4LG3-4 fusion protein was successfully expressed.更多还原