遗传 ›› 2003, Vol. 25 ›› Issue (2): 141-144.

• 论文 • 上一篇    下一篇

pET28a-TAT-LacZ重组子的构建

严世荣1;2;龚坚2;严洁3;邱云城1   

  1. 1.郧阳医学院附属十堰市人民医院检验部,湖北 十堰 442000;2.郧阳医学院生物化学教研室,湖北 十堰 442000;3.郧阳医学院附属太和医院神经内科,湖北 十堰 4420001.
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2003-04-10 发布日期:2003-04-10

Constructing the recombinant of pET28a-TAT-LacZ

YAN Shi-Rong1,2;GONG Jian2;YAN Jie3;QIU Yun-Cheng1   

  1. Department of Clinical laboratory,Shiyan Peoples Hospital of Yunyang medical college,Shiyan,Hubei 442000,China;2.Department of Biochemistry Yunyang Medical College,Shiyan,Hubei 442000,China;3.Taihe Hospital of Yunyang Medical College,Shiyan,Hubei 442000,China
  • Received:1900-01-01 Revised:1900-01-01 Online:2003-04-10 Published:2003-04-10

摘要: 为了构建高表达pET28a-TAT-LacZ重组子,观察表达的融合蛋白TAT-β-Gal能否穿过生物膜,使用人工合成编码TAT蛋白转导区的DNA片段,插入载体pET28a组氨酸编码区后再连接lacZ基因,组成pET28a-TAT-LacZ重组表达子,转化大肠杆菌,利用组氨酸亲和层析柱纯化TAT-β-Gal融合蛋白,将融合蛋白加入培养的平滑肌细胞。得到高度纯化的、有活性的TAT-β-Gal融合蛋白, TAT-β-Gal在短时间内进入体外培养平滑肌细胞,成功地构建了高表达pET28a-TAT-LacZ重组子,并在体外培养的细胞中证实TAT-β-Gal融合蛋白穿透生物膜的能力,为肽类、生物大分子药物进入组织细胞内发挥治疗作用提供了理论基础。

关键词: 平滑肌细胞, TAT-β-Gal融合蛋白, TAT蛋白转导区, pET28a质粒

Abstract: A synthesized double-stranded oligomeric nucleotide encoding 11-amino acid TAT protein transduction domain3 was inserted into pET28a vector after 6 histidind coding sequence,LacZ gene from pcDNA4/Myc-His/LacZ was digested by EcoRⅠand HindⅢ,then cloned into pET28a-TAT.The highly expressed TAT-β-galactosidase was purified by affinity chromatography.TAT-β-Gal fusion protein can across vascular smooth muscle cells in vitro.This result open new possibility for direcy delivery of protein into tissues for therapy.