GST-p43/AIMP1融合蛋白表达和纯化以及与NF-L的相互作用

目的 构建人p43/AIMP1蛋白与谷胱甘肽转移酶(GST)重组的融合蛋白原核表达载体并进行表达纯化,通过GST-pull down方法验证其与神经中间丝轻链(NF-L)的体外直接相互作用.方法 以重组质粒pcDNA3.1-p43为模板,扩增p43/AIMP1基因,产物经纯化回收后与原核表达载体pGEX4T-1连接构建成新载体GST-p43/AIMP1,经鉴定完全正确后转化大肠埃希菌B.21(DE3),通过异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达,并纯化获得目的蛋白；将myc-NF-L体外转染HEK293T细胞,利用GST pull-down原理和方法验证人p43蛋白与神经中间丝轻链蛋白NF-L之间的相互作用.结果 酶切鉴定和测序结果显示,成功构建了GST-p43/AIMPI融合蛋白原核表达载体；考马斯亮蓝染色和Western blotting结果显示,成功获得有生物活性的GST-p43/AIMPI融合蛋白；GST pull-down实验结果证实,p43/AIMP1与NF-L存在直接相互作用.结论 获得有生物活性的GST-p43/AIMP1蛋白,并成功应用GST pull-down方法证实p43/AIMP1与NF-L在体外存在直接的相互作用.

Expression and purification of GST-p43/AIMP1 fusion protein and its interaction with NF-L

Objective To construct and purify the prokaryotic expression vector of fusion protein of human p43/AIMP1 protein and glutathione-S-transferase(GST),and verify its direct interaction with neurofilament light subunit(NF-L) in vitro through GST-pull down assay.Methods p43/AIMP1 gene was amplified from pcDNA3.1-p43,and was inserted into prokaryotic expression vector pGEX4T-1 to generate novel vector GST-p43/AIMP1.After identification of GST-p43/AIMP1,Escherichia coli BL21(DE3) was transfected,which was induced by isopropyl-β-D-thiogalactoside(IPTG),and target protein was obtained after purification.HEK293T cells were transfected in vitro with myc-NF-L,and the interaction between GST-p43/AIMP1 and myc-NF-L was detected using GST pull-down assay.Results Restriction enzyme digestion and sequencing indicated that prokaryotic expression vector of GST-p43/AIMP1 fusion protein was successfully constructed.Coomassie brilliant blue staining and Western blotting revealed that GST-p43/AIMP1 fusion protein with bioactivity was successfully obtained.GST pull-down assay verified that there was direct interaction between p43/AIMP1 and NF-L.Conclusion The fusion protein of GST-p43/AIMP1 with bioactivity has been successfully obtained,and the direct interaction between p43/AIMP1 and NF-L has been verified in vitro through GST pull-down assay.