PTD-p53 融合蛋白的表达、纯化及其对肝癌细胞的转导活性-东南大学学报医学版

PTD-p53 融合蛋白的表达、纯化及其对肝癌细胞的转导活性

单位：1.南京医科大学附属无锡市第一人民医院,肝胆外科,江苏,无锡,214002； 2.南京医科大学附属无锡市第一人民医院,中心实验室,江苏,无锡,214002； 3.南京医科大学附属江苏省人民医院,肝脏外科,江苏,南京,210029

分类号：R735.7

出版年·卷·期（页码）：2007·26·第三期（194-197）

摘要：

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.

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.

参考文献：

[1] KUO P L, CHIANG L G, LIN C C. Resveratrol-induced apoptosis is mediated by p53-dependent pathway in HepG2 cells. 2002(1). doi:10.1016/S0024-3205(2)02177-X
[2] 梁英民, 蒋珊珊, 韩骅. TAT PTD_BCR/ABL SH3融合蛋白对白血病系K562凋亡诱导作用. 第四军医大学学报2002(9)
[3] SUN B, WU Z, RUAN Y. P21WAF1/Cip1 gene expression in primary human hepatocelluar carrinoma and its relationship with p53 gene mutation, 1999(1)
[4] 曹基辉, 张静, 翟成凯. 乙型肝炎病毒x基因与肝细胞癌. 东南大学学报(医学版)2003(3). doi:10.3969/j.issn.1671-6264.2003.03.021
[5] VIVES E, BRODIN P, LEBLEU B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cells nucleus. 1997(25)
[6] KUNNIHISA M, HIDEKI O. Inhibition of growth of human prostate cancer xenograft by transfection of p53 gene:gene transfer by electroporation, 2002(1)
[7] RAMESH R, SAEKI T, TENPLETON N. Successful treatment of primary and dissemindted human lung cancer by systemic delivery of tumour suppressor genes using an improved liposome vector, 2001
[8] HAGIVARA K, SAIJOY Y. Perspect on postgenome medicine:gene therapy for long cancer, 2001
[9] WARREN R S, KIM D H. Liver-directed viral therapy for p53-targeted adenoviruses and beyond, 2002
[10] GAUTAM A, DENSMORE C L, MELTON S. Aerosol delivery of PEI-p53 complexes inhibits B16-F10 lung mestases throuth regulation of angiogenesis. 2001(1). doi:10.1038/sj.cgt.7700405
[11] SHI M, WANG F S, WU Z Z. Synergetic anticancer effect of combined-type p53,GM_CSF and B7-1 genes on hepatocelluar carinoma cells in vitro. World Journal of Gastroenterology2003(1)

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录