Tat-p53融合蛋白的表达、纯化及其转导活性

目的:原核表达野生型p53与TatPTD(proteintransductiondomain)的融合蛋白,检测TatPTD介导p53进入肝细胞的效率。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pTAT-HA和pET-32a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Tat-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Tat-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Tat-p53融合蛋白及p53蛋白,制备p53特异的抗血清,证实Tat-p53可以高效的转入HepG2细胞内。结论:Tat-p53融合蛋白的表达纯化及活性分析,为应用Tat-p53融合蛋白治疗肝癌的实验研究奠定了基础。

Expression, purification and transduction of Tat-p53 fusion protein

AIM: 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. CONCLUSION: The obtained Tat-p53 fusion protein may be valuable for the basic research on therapy for liver carcinoma.