Cloning and Expression of p53 Gene and its Activity Assay
- VernacularTitle:p53基因的原核表达与活性测定
- Author:
Chunyan LI
;
Bing YANG
;
Xingyu LI
;
Yadong YU
- Publication Type:Journal Article
- Keywords:
p53 gene;
K562;
FCM;
Apoptosis
- From:
Journal of Medical Research
2006;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express and purify pET-p53 fusion protein and investigate the effects of the protein on the proliferation of human leukemia cell line K562. Methods The fragment of human wild type p53 cDNA was amplified by PCR and the expression plasmid of pET-p53 was constructed. Recombinant plasmids were transformed into E.coli BL21,then induced by IPTG at 1mmol/L. The protein was purified by the column of Ni-NAT and analyzed by SDS-PAGE. After treated with purified P53 protein with different concentrations,the proliferation of K562 was tested by MTT assay, clone formation and FCM. Results Prokaryotic expression vectors of pET-p53 were constructed correctly. pET-p53 fusion protein was successfully expressed and purified. With its increasing concentrations, P53 protein reversed its effect on K562 significantly. The ratio of inhibition had linear relation to the concentration of P53 protein when the concentration of the protein was between 0.1?g/ml and 100?g/ml. And its IC50 was 6.74?g/ml. Conclusion The obtained pET-p53 fusion protein might induce the leukemia cell line K562 apoptosis.
