p53基因的克隆、原核表达及功能初步研究
摘要
目的原核表达P53重组蛋白,检测P53重组蛋白对人类白血病细胞系K562和人肝癌细胞系SMMC-7721体外增殖的影响。方法运用PCR技术鉴定了pET-1053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。以不同浓度经磁性荧光纳米粒子(FMNP)修饰后的P53重组蛋白诱导K562细胞和SMMC-7721细胞,用MTT比色法、集落形成法、流式细胞术(FCM)测定P53蛋白对靶细胞增殖的影响。利用激光共聚焦显微镜(LSCM)观察经磁性荧光纳米粒子修饰的P53重组蛋白在细胞中的定位。结果1.成功的构建了含人野生型p53基因的原核表达载体,分离纯化了P53重组蛋白。2.磁性荧光纳米粒子修饰的P53重组蛋白较未修饰的蛋白有更好的凋亡诱导作用,且磁性纳米粒子本身并不具细胞毒害作用;3.P53重组蛋白抑制K562肿瘤细胞的生成,随药物剂量增大,细胞凋亡率上升,其半数抑制浓度(IC50)为6.74μg/ml 4.随着P53重组蛋白浓度的增加,SMMC-7721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,其半数抑制浓度(IC50)为12.39μg/ml 5.激光共聚集显微镜观察发现P53重组蛋白主要定位于细胞核,胞浆也有少量分布。结论原核表达的P53重组蛋白具有野生型P53(wtp53)蛋白活性,体外实验发现经磁性荧光纳米粒子修饰的P53重组蛋白能够抑制K562细胞和SMMC-7721细胞生长并促使它们发生凋亡。
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 and Human hepatocellular carcinoma cell line SMMC-7721 in vitro. 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 fluorescent magnetic nanoparticles(FMNP) modified P53 protein with different concentrations, the proliferation of K562 were tested by MTT assay, clone formation and FCM. We also used laser scanning Confocal microscope (LSCM) to observe the distribution of fluorescent magnetic nanoparticles modified P53 fusion protein in cells. Results Prokaryotic expression vectors of pET- p53 were constructed correctly. pET- p53 fusion protein were successfully expressed and purified. With its increasing concentrations, modified P53 fusion protein reversed its effect on tumor cells significantly. The ratio of inhibition was linear relation to the concentration of P53 fusion protein when concentration of the protein was between 0.1μg/ml and 100μg/ml. Its IC50 was 6.74μg/ml in K562 cells and was 12.39μg/ml in SMMC-7721 cells. Laser scanning Confocal microscope to observe the distribution found that the protein mainly distributed in the nucleus and a small portion of protein was in the cytoplasm. Conclusions The obtained pET- p53 fusion protein has the biological activity of innate P53 protein and it might induce Human leukemia cell line K562 and Human hepatocellular carcinoma cell line SMMC-7721 apoptosis in vitro.
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 and Human hepatocellular carcinoma cell line SMMC-7721 in vitro. 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 fluorescent magnetic nanoparticles(FMNP) modified P53 protein with different concentrations, the proliferation of K562 were tested by MTT assay, clone formation and FCM. We also used laser scanning Confocal microscope (LSCM) to observe the distribution of fluorescent magnetic nanoparticles modified P53 fusion protein in cells. Results Prokaryotic expression vectors of pET- p53 were constructed correctly. pET- p53 fusion protein were successfully expressed and purified. With its increasing concentrations, modified P53 fusion protein reversed its effect on tumor cells significantly. The ratio of inhibition was linear relation to the concentration of P53 fusion protein when concentration of the protein was between 0.1μg/ml and 100μg/ml. Its IC50 was 6.74μg/ml in K562 cells and was 12.39μg/ml in SMMC-7721 cells. Laser scanning Confocal microscope to observe the distribution found that the protein mainly distributed in the nucleus and a small portion of protein was in the cytoplasm. Conclusions The obtained pET- p53 fusion protein has the biological activity of innate P53 protein and it might induce Human leukemia cell line K562 and Human hepatocellular carcinoma cell line SMMC-7721 apoptosis in vitro.
引文
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