GST-NRP-1融合蛋白的原核表达及纯化

Prokaryotic expression and purification of GST-NRP-1 fusion protein

目的:构建带有GST标签的人NRP-1融合蛋白原核表达载体,在大肠埃希菌(E.coli)中诱导其表达,并进行包涵体的复性及纯化。方法:利用RT-PCR扩增人NRP-1基因,将其克隆至p CR-blunt载体,酶切制备NRP-1基因片段,插入表达载体p GEX-4T-1,生成p GEX-4T-1-NRP-1,转入BL21感受态细胞以IPTG诱导其蛋白的表达。裂解细菌后行Western blot检测GST-NRP-1融合蛋白的表达,表达的包涵体经复性后用Glutathione Sepharose 4B纯化。结果:RT-PCR扩增出NRP-1基因并连入p CR-blunt载体;经亚克隆构建了融合蛋白表达载体p GEX-4T-1-NRP-1。转入BL21感受态细胞后考马斯亮蓝染色检测到GST-NRP-1融合蛋白的表达,经包涵体复性后得到纯化的融合蛋白。结论:成功构建带有GST标签的人NRP-1融合蛋白原核表达载体,为进一步研究NRP-1的结构、功能及其与之相互作用的蛋白奠定了基础。

Objective：To construct GST-tagged human NRP-1 fusion protein expression vector and induce its expression in Escherichia coli （ E.coli） ,then carry on inclusion body refolding and purification so as to obtain GST-NRP-1 fusion protein.Methods：NRP-1 gene was amplified by RT-PCR and inserted into pCR-blunt vector.Then the reconstructed plasmid was inserted into prokaryotic expression vector pGEX-4T-1.The constructed pGEX-4T-1-NRP-1 expression vector was transformed into BL21 cells and induced by i-sopropyl-β-D-thiogalactoside （ IPTG）.Bacterial bodies were disrupted by sonication.Then the soluble fraction of fusion proteins were verified by Western blot and purified by Glutathione Sepharose 4B after inclusion body refolding.Results： The NRP-1 gene fragment was amplified by RT-PCR and inserted into pCR-blunt vector.Fusion protein expression vector pGEX-4T-1-NRP-1 was constructed suc-cessfully.After transformation, GST-NRP-1 expression vector was detected in BL21 cells and obtained purifying protein after refolding.Conclusion：The plasmid GST-NRP-1 was constructed successfully and laid basis for subsequent studies.