【作者】 陈春；

【导师】 詹金彪；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 [目的] 蓖麻毒素（Ricin）是一种从蓖麻籽中分离到的毒蛋白,由A链（RTA）和B链（RTB）组成,两条链通过二硫键连接。RTA由236个氨基酸组成,分子量约32kD,RTA第10个残基Asn为糖基化部位,天然的毒素分子接有（GlcNAc）₂（Man）₄寡糖链。活性保守位点Glu177和Arg180位于催化活性裂隙,他们对RTA的酶活性至关重要。RTA相当于Ⅰ型核糖体失活蛋白（RIP）,具有N-糖苷酶活性,可以裂解真核细胞核糖体的28S RNA上4234位腺苷酸的N-糖苷键;RTB可识别末端含半乳糖的受体,协助RTA链进入细胞内。蓖麻毒素的作用过程一般是这样的:先是RTB与细胞膜受体结合,主要经过受体介导的内吞过程进入吞噬体,然后沿内体、高尔基体、内质网等逆向分泌途径,有序地运输到内质网,RTA最后从内质网转位进入细胞浆,在胞浆内攻击核糖体,从而抑制细胞蛋白质的合成,导致细胞死亡。一般生理状况下,一分子的RTA每分钟能催化脱去1000-2000个哺乳动物细胞的腺嘌呤,使核糖体失去功能。RTA的细胞毒性作用,可被用来与单克隆抗体或细胞因子结合形成导向药物—免疫毒素,用于肿瘤的靶向治疗,还可用来治疗移植物抗宿主反应和自身免疫性疾病等。 近年来,有关蓖麻毒素等RIP的作用机理研究,大多集中在它们在胞内的运输和转位途径上。目前较为一致的看法是,毒素分子内吞后的逆向转运途径,可能有内体、高尔基体、溶酶体等细胞器的参与;但溶酶体是否参与毒素分子的运输和降解,至今没有定论。更多还原

【Abstract】 [Purposes]Ricin toxin (RT) is a plant toxin isolated from the seeds of Ricinus Communis. It is one of the most powerful plant toxins discovered. The toxin protein consists of two chains linked by a disulfide bond. Ricin A chain (RTA) is an active chain and has specific N-glycosidase activity, which excises a specific adenine residue (A4324 in rat) from a highly conserved loop of 26 or 28S rRNA in 60S ribosomal subunits. This cleavage of adenine can lead to the disruption of ribosomal function, thereby, inhibits the protein synthesis and then causes the death of cells. Ricin B chain (RTB) can specifically recognize receptors on the cell membrane, which have a terminal galactose. RTB is responsible for the binding to the cell receptors and may facilitate the translocation of RTA. Ricin enters the cells by receptor-mediated endocytosis, followed by translocation across the membranes of intracellular compartments.Ricin enters the cells by receptor-mediated endocytosis, followed by translocation across the membranes of intracellular organdies. In this study, a lysosomes retention signal KFERQ was fused to the C-terminus of ricin (RTA) by polymerase chain reaction. The recombinant RTA and RTA-KFERQ were expressed in Escherichia coli using plasmid pKK223.3 under the control of a tac promoter. The recombinant proteins were purified by affinity chromatography on a Blue-Sepharose 6B column. The cytotoxicities of RTA and the fusion toxin RTA-KFERQ were measured by the MTT assay in HEPG2, Hela, and A549 cells following fluid-phase endocytosis.[Methods]1. Expression and purification of recombinant RTAsThe expression plasmid pKK223.3-RTA was introduced into E. coli JM 109 by CaCb-medicated method. The cells were grown until an optical density at 600 nm reached 0.6. Expression was induced in the presence of IPTG to a final concentration of 1 mM and incubation with shaking at 30°C for 3 h. In order to evaluate the effect of temperature on expression of rRTAs, we have investigated the distribution of the recombinant proteins expressed at different temperatures. To identify an optimal length of the incubation, the bacteria were induced at 30°C for 1 h, 3 h and 5 h respectively. The distribution of the recombinant proteins was analyzed on 12% SDS-PAGE. After induced by 0.1 M IPTG, the pellet was resuspended in phosphate-buffered saline (PBS)/5 mM EDTA, and sonicated three times for 20 s at maximal output. Debris was removed by centrifugation at 15000 rpm for 15 min at 4℃. The supernatants were dialyzed against 50 mM phosphate buffer (PB) (pH 6.5), and recombinant RTAs (rRTAs) were affinitively purified on a Blue-Sepharose 6B column by elution with 0.65 M sodium chloride. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to check the purity. The fractions containing the rRTAs were collected, and the protein concentration was measured by Bradford method.2. Cytotoxicity AssayHEPG2, Hela and A549 cells were planted out in 96-well plates at a density of 1.2 x 10~4 cells per well in the serum-free RPMI-1640 medium. Various concentrations of rRTA and rRTA-KFERQ were added into the wells and incubated for 8 h to allow internalization of toxins by fluid-phase endocytosis. The cells were then incubated in RPMI-1640 medium containing 10% fetal calf serum for another 48 h. Cell viability was measured by MTT assay. In this assay, trtrazolium salt (MTT) is converted to a blue formazan product by enzymes active only in living cells. The amount of formazan produced can be quantitated using a microtitre plate reader. In order to get precise data, each concentration was set for 5 wells.[Results]1. Expression of recombinant RTA and RTA-KFERQExpression of rRTA and rRTA-KFERQ was induced with 0.1 mM IPTG at 30℃ for 3 h. SDS-PAGE showed that the target proteins with approximate molecular weight, 31 kD could be detected in the lysates of the bacteria. The rRTA induced at 30℃ for 3h was soluble and fully active. In comparison to rRTA, a major part of rRTA-KFERQ was expressed in a soluble style at 30℃ for 3h, whereas that expressed更多还原