【作者】 王小波；

【导师】 屈伸；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 1988年Green和Frankel各自独立发现HIV-TAT( human immunodeficiency virus trans-activator of transcription,HIV-TAT)蛋白具有穿透生物膜的能力,该蛋白全长86个氨基酸。TAT蛋白具有穿透生物膜的区域称为蛋白转导域(Protein transduction domain, PTD),由11个氨基酸组成,研究表明TAT是一个极好的蛋白质、多肽及基因的转运载体。TAT的11个氨基酸转导序列是目前为止所发现的最短的蛋白质转导序列。细胞凋亡是机体清除多余、变异或肿瘤细胞的一种主动、程序化的生理过程。该过程的紊乱与肿瘤的发生密切相关。近年来,病毒基因产物对细胞凋亡的调节作用逐渐引起人们的重视。鸡贫血病毒(chicken anemia virus, CAV)就是一种诱导细胞凋亡的新病毒。VP3蛋白(Apoptin,亦称凋亡素)源于鸡贫血病毒,由121个氨基酸组成,能够特异性诱导肿瘤细胞的凋亡,令人感兴趣的是VP3对正常的人和鼠的二倍体细胞无任何毒副作用,因此VP3在抗肿瘤方面具有巨大的潜在优势和广阔的应用前景。本研究通过分子克隆常规方法构建了TAT-VP3、TAT-GFP及TAT-GFP-VP3等3种融合蛋白的pET28a原核表达载体,IPTG诱导融合蛋白表达,经镍金属鳌合层析柱纯化获得融合蛋白。通过蛋白转导实验观察其透膜效应,并观察融合蛋白对肿瘤细胞凋亡的诱导效应。结果显示:通过荧光显微镜观察,发现融合蛋白TAT-GFP及TAT-GFP-VP3能进入体外培养的细胞,在荧光显微镜下可见细胞内有绿色荧光。融合蛋白TAT-VP3及TAT-GFP-VP3可诱导体外培养的HepG2细胞凋亡。更多还原

【Abstract】 In 1988,Green and Frankel found respectively that protein HIV-TAT has the function of permeating into cells through membrane. Protein TAT consists of 86 amino acids in its full length. The domain which has transmembrane function is named as protein transduction domain (PTD). It consists of 11 amino acids. The research indicated that PTD is a wonderful transduction vector for protein, polypeptide even gene. The sequences of PTD with 11 amino acids is the shortest protein transduction domain by far.Apoptosis is an initiative programmed physiological process for clearing excrescent, abberance or tumor cells. Disorder of this process was found closely relation to the development of tumor. Recently, the regulating roles in cells by products of virus gene CAV(chicken anemia virus) is a new virus which can induce the apoptosis of tumor cells. The chicken anemia virus (CAV)-derived protein VP3 (Apoptin) which consists of 121 amino acids has been shown to induce apoptosis in various tumor cells, but not in normal human cells and murine diploid cells which interest us mostly. Apoptin is a powerful apoptosis-inducing protein with a potential for cancer therapy.The research is based on the constructing of the prokaryotic expression recombinants of pET28a-TAT-vp3, pET28a-TAT-GFP and pET28a-TAT-GFP- vp3 by routine molecular cloning. The expression of three kinds of fusion proteins were induced by IPTG and the purified protein was obtained by nickel affinity gel column chromatography. The transmembrane function and the induced-apoptosis effects on tumor cells were observed by protein transduction assay. The result showed that fusion proteins TAT-GFP and TAT-GFP-VP3 can permeating into cells cultured in vitro and green fluorescences can be found under fluorescence microscope. Fusion proteins TAT-VP3 and TAT-GFP-VP3 could induce apoptosis of HepG2 cells cultured in vitro.更多还原