[Expression, purification and transduction of Tat-p53 fusion protein].
OBJECTIVE
To express and purify Tat-p53 fusion protein and investigate its transduction efficiency.


METHODS
The gene encoding wide-type p53 was isolated using RT-PCR from A549 cell line and cloned into pTAT-HA and pET32a prokaryotic expression vectors. Recombinant plasmids were transformed into E.coli BL21(DE3)LysS, then the transformed cells were induced with IPTG. The expression and purification of the Tat-p53 and p53 were analyzed by SDS-PAGE. BALB/c mice were immunized with purified p53 protein. The serum was isolated and the antibody specific to p53 was measured by ELISA. The transduction efficiency of Tat-p53 was detected using indirect immunofluorescence assay.


RESULTS
Prokaryotic expression vectors of Tat-p53 and p53 were constructed correctly. Tat-p53 fusion protein and p53 protein were successfully expressed and purified. p53 specific mouse antiserum was obtained. IFA result indicated that Tat-p53 fusion protein transduced into HepG2 cells efficiently.


CONCLUSIONS
The obtained Tat-p53 fusion protein may be valuable for the basic research on therapy for liver carcinoma.
Ding
Jin
J
Department of Etiology, Fourth Military Medical University, Xi'an 710032, China. dingjin1103@sina.com
Liu
Jun
J
Huang
Yu-xiao
YX
Li
Ying-hui
YH
Chen
Jun
J
Zhao
Ya
Y
Xue
Cai-fang
CF
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D015696:Gene Products, tat / Q000235:genetics*; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D015696:Gene Products, tat; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53