title: [Reconstruction of an intracellular transduction system based on HIV-1 TAT protein transduction domain].
abstract: OBJECTIVE
  To explore the reasons for the low intracellular transduction efficiency of a previously constructed His-TAT-Flag recombinant protein and establish a more efficient transduction system.
  
  
  METHODS
  The Flag tag of pET14b-His- Tat-Flag vector was deleted with PCR mutant kit, and enhanced green fluorescent protein (EGFP) coding sequence was inserted into the new pET14b-His-TAT recombinant vector. Enzyme digestion and DNA sequencing were performed for identification of pET14b-His-TAT-EGFP vector, which was then transformed into E. coli BL21(DE(3)). After IPTG induction, the recombinant protein of His-TAT-EGFP was isolated and analyzed with SDS-PAGE. Purified His-TAT-EGFP recombinant protein was added to ECV304 cells and the fluorescence was observed to evaluate the transduction efficiency.
  
  
  RESULTS
  pET14b-His-TAT vector and pET14b-His-TAT- EGFP vector were successfully constructed, which was identified with enzyme digestion and DNA sequencing. His-TAT-EGFP fusion protein was expressed and purified successfully and showed cellular transduction activity.
  
  
  CONCLUSIONS
  The prokaryotic expression vector has been successfully constructed by modifying pET14b-His-TAT-Flag, and the expressed and purified recombinant protein of His-TAT-EGFP possesses high efficiency of intracellular transduction activity.
authors.affiliation: Department of Pathophysiology/Key Laboratory of Functional Proteomics of Guangdong Province, Southern Medical University, Guangzhou 510515, China.
authors.forename: Ai-hua
authors.initials: AH
authors.lastname: Guo
authors.forename: Zhi-feng
authors.initials: ZF
authors.lastname: Liu
authors.forename: Xue-gang
authors.initials: XG
authors.lastname: Sun
authors.forename: Hai-yu
authors.initials: HY
authors.lastname: Li
authors.forename: Peng
authors.initials: P
authors.lastname: Deng
authors.forename: Yong
authors.initials: Y
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins