TAT-NDPK-A蛋白的构建表达及穿膜初步研究  

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

作　　者：刘秋英[1] 王一飞[1] 熊盛[1] 张美英[1] 钱垂文[1] 何旭君 

机构地区：[1]暨南大学生物医药研究开发基地,广州510632 [2]广东省林业学校,广州510520

出　　处：《中国药学杂志》2007年第9期652-655,共4页Chinese Pharmaceutical Journal

基　　金：国家自然科学基金面上项目(30400071);广东省科技计划项目(2005B50301017);人事部留学人员科技活动项目(国人厅发[2005]129)

摘　　要：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。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 Ni^2＋ -NTA affinity column. The antigenicity of fusion protein was analyzed by westen-blot. Fusion protein was added to cultured A549 cells and was ob- served 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.

关 键 词：TAT 核苷二磷酸激酶A蛋白 融合蛋白 

分 类 号：R915.2[医药卫生—微生物与生化药学]