TAT-NDPK-A蛋白的构建表达及穿膜初步研究

 目的构建pET-TAT-nm23-H1质粒,表达TAT-核苷二磷酸激酶A(TAT-nucleoside diphosphate kinase A,TAT-NDPK-A)融合蛋白,并研究融合蛋白穿膜进入A549细胞。方法人工合成编码TAT蛋白转导区域的11个氨基酸序列于NDPK-A引物的上游,PCR扩增后将融合基因克隆到pET28(a)原核表达载体上,经IPTG诱导表达、镍离子树脂色谱纯化TAT-NDPK-A蛋白,Westen-blot鉴定分析重组蛋白的抗原性,将TAT-NDPK-A蛋白加入到A549细胞中,荧光免疫组化检测蛋白穿膜进入细胞内。结果TAT序列与NDPK-A正确融合并插入pET28(a)载体上,经诱导表达纯化成功获得纯度为97%的TAT-NDPK-A蛋白,在培养的A549细胞中加入重组蛋白4 h后在细胞内检测到穿膜的蛋白。结论构建表达TAT-NDPK-A蛋白,TAT序列能够引导重组蛋白穿过细胞膜进入细胞内,为进一步研究基因的功能奠定了基础。

Construction and Expression of TAT-NDPK-A Protein and Its Penetration into the Cells

OBJECTIVE To construct the plasmid of pET-TAT-nm23-H1,express the fusion protein of TAT-NDPK-A,and study the penetration of fusion protein into A549 cells.METHODS The fused gene sequences of TAT and nm23-H1 were obtained after PCR with the upstream primer including the DNA sequence of TAT,and then cloned into pET28(a) vector.Recombinant plasmid was sequenced and transformed to Escherichia coli BL21.TAT-NDPK-A was expressed after IPTG induction and purified by Ni2+-NTA affinity column.The antigenicity of fusion protein was analyzed by westen-blot.Fusion protein was added to cultured A549 cells and was observed by fluorescence immunochistochemistry.RESULTS TAT-nm23-H1 was cloned into pET28(a) vector correctly.The purificaton of TAT-NDPK-A protein was 97% after expression induced by IPTG and purification.TAT-NDPK-A protein was delivered into A549 cells by penetrating the cell membrane.CONCLUSION The fusion protein of TAT-NDPK-A is successfully expressed and purified.TAT sequences can deliver the fusion protein to penetrate the cell membrane and enter the cells.It lays solid foundation for the further research on gene function.