PTD-p53 融合蛋白的表达、纯化及其对肝癌细胞的转导活性

目的：原核表达野生型p53与PTD的融合蛋白,检测PTD介导p53进入肝癌细胞的效率。方法：利用RT-PCR方法从A549细胞系中分离野生型p53基因,将该基因分别克隆入pTATHA和pET32a原核表达载体,在大肠杆菌BL21（DE3）LysS内诱导表达并进行纯化。以纯化的p53蛋白经腹腔免疫BALB/c小鼠,制备高效价的抗血清。将PTD-p53融合蛋白加入HepG2细胞培养上清,利用间接免疫荧光法检测PTD-p53融合蛋白导入HepG2细胞的效率。结果：成功地构建了含有野生型p53基因的原核表达载体,表达纯化了p53融合蛋白及p53蛋白,证实PTD-p53可以高效地转入HepG2细胞。结论：PTD-p53融合蛋白的表达纯化及活性分析,为应用PTD-p53蛋白治疗肝癌的实验研究奠定了理论基础。

Expression,purification and transduction of PTD-p53 fusion protein to hepatocellular carcinoma cell

Objective To express and purify PTD-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 pTATHA and pET32a prokaryotic expression vectors.Recombinant plasmids were E.coli BL21(DE3)LysS,then the transformed cells were induced with IPTG.The expression and purification of the PTD-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 PTD-p53 was detected using indirect immunoflure scence assay.Results Prokaryotic expression vectors of PTD-p53 and p53 were constructed correctly.PTD-p53 fusion protein and p53 protein were successfully expressed and purified.p53 specific mouse antiserum was obtained.IFA result indicated that PTD-p53 fusion protein transduced into HepG2 cells efficiently.Conclusion The obtained Tat-p53 fusion protein provides a theretical foundation for the basic research on PTD-p53 treating hepatocellular carcinoma.