Reconstruction of an intracellular transduction system based on HIV-1 TAT protein transduction domain / 南方医科大学学报
Journal of Southern Medical University
;
(12): 545-548, 2006.
Article
in Chinese
| WPRIM
| ID: wpr-255258
ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Recombinant Fusion Proteins
/
Molecular Sequence Data
/
Transfection
/
Cell Line
/
Amino Acid Sequence
/
HIV-1
/
Protein Transport
/
Green Fluorescent Proteins
/
Electrophoresis, Polyacrylamide Gel
/
Tat Gene Products, Human Immunodeficiency Virus
Type of study:
Prognostic study
Limits:
Humans
Language:
Chinese
Journal:
Journal of Southern Medical University
Year:
2006
Type:
Article
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