p53基因的原核表达与活性测定  

Cloning and Expression of p53 Gene and its Activity Assay

作　　者：李春炎[1] 杨兵[1] 李兴玉[1] 俞亚东[1] 

机构地区：[1]上海师范大学生命与环境科学学院生物系,200234

出　　处：《医学研究杂志》2009年第1期38-41,共4页Journal of Medical Research

摘　　要：目的原核表达P53重组蛋白,检测P53蛋白对人类白血病细胞系K562增生的影响。方法运用PCR技术鉴定了pET-p53重组质粒导入BL21菌株中p53基因的存在,并在IPTG的诱导下,大量产生外源基因产物。通过Ni-NAT亲和层析柱纯化分离P53蛋白,SDS-PAGE确定该蛋白准确性。以不同浓度的P53重组蛋白诱导K562细胞后,用MTT比色法、集落形成法、流式细胞术(FCM)测定P53蛋白对靶细胞增生的影响。结果成功地构建了含人野生型p53基因的原核表达载体,纯化了P53重组蛋白。活性测定结果表明,随着P53重组蛋白浓度的增加,K562细胞存活率显著降低,并发现该蛋白能促进肿瘤细胞凋亡,且呈现明显的剂量依赖性,经相关分析,细胞抑制率与P53蛋白浓度呈正相关(r=0.8981),其半数抑制浓度(IC50)为6.74μg/ml。结论P53重组蛋白具有促进K562细胞凋亡的作用。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 eDNA was amplified by PCR and the expression plas- mid of pET -p53 was constructed. Recombinant plasmids were transformed into E. eoli BL2I ,then induced by IPTG at lmmol/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 Prokaryotie 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 μg/ml and 100μg/ml. And its IC50 was 6. 74μg/ml. Conclusion The obtained pET - p53 fusion protein might induce the leukemia cell line K562 apoptosis.

关 键 词：P53基因 K562 FCM 细胞凋亡 

分 类 号：R735.7[医药卫生—肿瘤] R394[医药卫生—临床医学]