【作者】 陈兴勇；

【导师】 徐康森； 刘兢；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 蛇毒主成份为具有多种生物活性的蛋白和肽类的混合物。一些蛋白作为神经毒素、细胞毒素和组织坏死性诱导剂而具有致死效应,另外一些可引起多种不同的药理学反应,且毒性较低。所有这些蛋白毒素均可攻击细胞上某些特定的位点而影响生理进程。中华眼镜蛇细胞毒素（Cardiotoxin,CTXs）为一类可与细胞膜作用的碱性多肽,主要通过结合于细胞膜表面磷脂或膜蛋白引发多种药理效应。近年来,纯化的各种眼镜蛇细胞毒组分已广泛用于实体瘤和血液病的治疗研究。本论文旨在研究细胞毒素组分三（CTXⅢ）的克隆、表达和纯化,重组细胞毒素活性分析以及细胞毒素作为临床抗肿瘤药物的可行性研究。主要包括以下三部分内容:1、研究了CTXⅢ的理化性质,并测定了N端15个氨基酸残基序列,克隆得到CTXⅢ基因。将其分别装入两种原核E.coli表达载体和真核酵母表达载体中进行表达。CTXⅢ在pET30a载体中表达时,在胞内形成包涵体,以不可溶的形式存在。CTXⅢ基因插入pThioHis B载体中,与硫氧还蛋白（Trx）融合表达,通过肠激酶位点裂解可将融合蛋白释放出来。在实验室规模,每升培养液可表达7mg重组心脏毒素,经Histrap柱纯化回收,得率可达75%,肠激酶裂解后凝胶柱回收率为70%,总回收率达37%左右。CTXⅢ基因插入真核酵母分泌型表达载体（PIC9K）,经诱导重组蛋白表达量为9.5mg/L,培养液经离心后直接凝胶柱回收重组目的蛋白,得率可达65%。与天然毒素相比,重组蛋白N端冗余三个氨基酸（Gly-Tyr-Thr）,RP-HPLC分析重组蛋白纯度大于95%。利用溶血活力和对K562肿瘤细胞增殖抑制检测重组细胞毒素与天然细胞毒素的活性,溶血分数50%时所对应的质量浓度（HU50）分别为6.53μg/ml和7.4μg/ml,12小时IC50分别为3.56μg/ml和2.63μg/ml,表明重组蛋白活性接近天然蛋白,推测分泌表达时重组蛋白折叠正确。选择真核酵母分泌型表达载体为最佳表达方式。2、采用不同生物学方法分析细胞毒素体外作用于血液病细胞K562的杀伤效果,通过细胞增殖抑制、Annexin V/PI双染实验、Caspase活性检测和DNA片段化等观察CTXⅢ诱导下K562细胞凋亡的情况。结果显示,CTXⅢ作用于K562细胞可使细胞停滞于G2/M期,并且活化Caspase-8和细胞色素C双重信号途径,诱导下游Bid分子切割,促使K562细胞发生凋亡。MAPK特异性阻断剂和免疫印迹分析发现,CTXⅢ诱导K562细胞凋亡的过程中,通过磷酸化p38和JNK蛋白,激活MAPK信号通路。建立鸡胚尿囊膜（CAM）体内K562肿瘤模型实验,高剂量CTXⅢ不影响鸡胚发育,表明CTXⅢ基本无毒副作用,CTXⅢ亦可有效抑制CAM上K562细胞形成肿瘤结节的增长,这一过程与Caspase-8和细胞色素C途径相关。通过分析K562血液病的形成机制,认为CTXⅢ可能是通过竞争性结合ATP,从而阻断K562细胞中异常酪氨酸激酶活性。3、本论文同时分析了CTXⅢ作用于实体瘤肝癌的应用效果,在较低剂量下,CTXⅢ可有效抑制HepG2细胞增殖(24小时的IC₅₀=2.58μg/ml),但对Bel-1402无明显作用。进一步研究发现,DAPI染色、Annexin V/PI双染实验和DNA片段分析证明CTXⅢ促使细胞发生凋亡,其凋亡过程受死亡受体途径和线粒体途径两种机制调控。CTXⅢ的作用早期HepG2细胞周期停滞于S期,半定量PCR和免疫印迹实验发现,周期素蛋白Cyclin D1、Cyclin A和Cyclin E在转录和翻译过程中,其蛋白和mRNA水平均有所下调,Bcl-2/Bax的蛋白和mRNA比例亦均发生变化。尾静脉注射CTXⅢ引起小鼠快速死亡,LD₅₀为936.7±38μg/kg。建立体内裸鼠HepG2肿瘤模型,经口服给药结果表明,CTXⅢ（2 mg/kg/d）可明显抑制肿瘤生长,其抑瘤率可达39.4%。病理检查心、肝、脾、肺、肾及小肠等脏器,未发现出血或坏死,表明CTXⅢ经口服对动物体基本无毒副作用。综上所述,CTXⅢ体内、外作用均可有效的抑制肿瘤细胞（K562、HepG2）的增殖,真核酵母表达产生的重组细胞毒素表现出与天然毒素类似的生物活性,因此,利用重组表达技术,不仅为蛇毒抗肿瘤药物提供来源,而且为研究三指蛋白的结构——功能关系提供了丰富的素材。更多还原

【Abstract】 The major components from snake venom are pharmaceutically complex of proteins and peptides which exert different biological activities.Some proteins induce death as neurotoxin,cardiotoxin or organic necrosis,while there are also some low toxic components which induce different pharmaceutical effects.These toxins affect normal physiological process through attack the specific sites on target cells. Cytotoxins from Naja naja atra are basic polypeptides which bind to susceptible cells on proteins or membrane phospholipids.In recent years,purified cobra snake venom components were widely applied on different solid tumor and leukemia study.In this article,we summarized the cloning,expression and purification of recombinant cytotoxinⅢ（CTXⅢ）,and the application as chemotherapeutic drug on anti-tumor therapy.There were three major parts in this dissertation.1.The physiological characteristics of native CTXⅢfrom cobra（Naja naja atra） were obtained by physical-chemical analysis,the CTXⅢhad a purity of more than 95%by RP-HPLC and TOF-MASS.The N-terminal sequence of CTXⅢwas determined by Ediman degradation method.The gene of CTXⅢwas obtained by RT-PCR method and was cloned into two prokaryotic expression vectors and eukaryotic yeast Pichia pastoris pPIC9K vector.The recombinant protein from pPEY30a was expressed as insoluble inclusion bodies.CTXⅢgene,fused with enterokinase in E.coli His-patch Thioredoxin expression system,was expressed in soluble form and released by osmotic-shock treatment.The recombinant CTXⅢwas released from fusion protein by enterokinase lysis.The production and purification process were performed at lab scale.About 7 mg recombinant CTXⅢwas obtained from per liter of bacteria culture with overall recovery of 37%.While the yield of recombinant CTXⅢfrom pichia pastoris pPIC9K was about 9.5 mg from per liter medium.Using straightforward one-step chromatography procedure,the rCTXⅢ, with three additional amino acids（GYT） at the N-terminal site,was purified to a purity of more than 95%and recovery yield of 65%.The purified rCTXⅢwas further characterized by hemolytic activity and cytotoxic assay compared with natural CTXⅢwith HD₅₀ 6.53μg/ml and7.4μg/ml,respectively,and IC₅₀ 3.56μg/ml and 2.63μg/ml,respectively,suggested that the activity of rCTXⅢwas similar to the nCTXⅢand the rCTXⅢhave the right conformation.The three additional amino acids（GYT） at the N termini of the rCTXⅢdid not affect the formation of disulfide bonds and cytotoxicity of the protein.The Eukaryotic vector was selected as the ideal expression form for CTXⅢ.2.Several methods including Annexin V/PI staining assay,Caspase activeity test and DNA fragmentation analysis,were used to evaluate the apoptosis of K562 cells induced by CTXⅢ.Data showed that CTXⅢinhibited proliferation of human leukemia K562 cells by G2/M phase arresting and apoptosis which was associated with the activation of caspase-8 and cytochrome c release,inducing the subsequent truncation of Bid.Western blotting detected the phosphorylatin of p38 MAPK and JNK when K562 cells were exposed to CTXⅢ.CTXⅢmediated apoptosis and phosphorylation of K562 cells were reduced by treatment with the MAPK-specific inhibitors indicated the activation of both JNK and p38 signaling pathway.A novel in vivo system-chicken chorioallantoic membrane-was developed for rapid assessment of leukemia k562 cell growth and treatment by chemotherapeutic CTXⅢ.Large doses of CTXⅢdid not infect the growth of embryo noted that CTXⅢhas little toxicity toward organism.Daily administration of CTXⅢfor two days to chicken chorioallantoic membrane（CAM） bearing tumours derived from the CAM at E10 administration of K562 cells resulted in inhibition of the tumours in vivo.Importantly, this in vivo inhibition was also associated with caspase-8 activation and cytochrome c release.Taken the formation of K562 cells into account,a hypothesis thus consider that CTXⅢcompetitively binding to ATP and block off the aberrant tyrosine kinase consecutive binding with ATP.These may thus explain the high efficacy of clinical treatment of CTXⅢ.3.CTXⅢon solid tumor therapeutic effects were next analyzed.In this experiment, we investigated whether CTXⅢaffects cell growth and cell cycle progression of hepatocellular carcinoma cells.We found that the proliferation of HepG2 cell was inhibited by CTXⅢ,to some extent,in a time- and dose-dependent manner（IC50 2.58 mg/ml at 24 h） but little effects on Bel-1402 cells.DAPI staining,flow cytometric analysis and annexin V labeling also demonstrated that CTXⅢincreased the percentage of apoptotic cells being associated with cell cycle arrest at S-phase. The apoptotic processs was associated with factor associated suicide and cytochrome c releasing.Semi-quantitative reverse transcription-polymerase chain reaction and western blot revealed that cyclin D1,cyclin A and cyclin E,which involved in cell apoptosis and cell cycle progression,were down regulated both at transcription and transcription levels.CTXⅢ-induced caspase-8,-9 and caspase-3 activation, generation of truncated Bid,releaseing of cytochrome c and the change of Bcl-2/Bax ratio on protein and mRNA levels.In vivo tests detected that CTXⅢwould cause sudden death when i.p.injected with LD50 936.7±38μg/kg.Tumor growth inhibition was most evident in mice treated with CTXⅢat 2 mg/kg/d,where an approximately 39.4%reduction in tumor size was observed,in contrast with mice treated with saline. No obvious toxicity,as judged by parallel monitoring of body weight and tissue sections of heart,intestine,liver and lung,was observed in CTXⅢ-treated mice.In conclusion,CTXⅢcould effectively inhibit the proliferation of tumor cells （K562 cells,HepG2 cells） both in vitro and in vivo.Recombinant CTXⅢfrom pichia pastoris has the similar biological activity compared to native CTXⅢ.More importantly,this protocol can be easily used for the production of the toxin at a larger scale with low cost.The approach outlined in this report will be suitable for the production of other recombinant proteins especially those of the ’three-finger’ family.更多还原