【作者】 王慧；

【导师】 张富春；

【作者基本信息】 新疆大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 细粒棘球蚴病又称囊型包虫病(Cystic echinococcosis,CE),是由细粒棘球绦虫(Echinococcus granulosus,Eg)幼虫引起的寄生虫病,是一种严重危害人类健康和畜牧业生产的人兽共患寄生虫病,呈全球性分布。目前对人的包虫病治疗以手术治疗为主,药物治疗为辅,但手术对人体损伤大且可能复发,服用阿苯哒唑和甲苯咪唑则可产生严重的不良反应,所以无法从根本上消灭包虫病。由于包虫病发病缓慢,易于传播,至今没有一种有效的预防措施,严重威胁牧区人民的健康和畜牧业发展。因此,对包虫病的早期诊断和免疫预防也就成为当前的研究热点。由于传统的病原学方法,诊断该病较困难,故常用影像学和免疫学方法作为辅助诊断手段,尤其免疫学方法以其费用低廉、操作简便、特异性高而成为目前包虫病诊断中的重要手段。但该病免疫学诊断因缺少标准、特异的抗原而影响了实验的敏感性和结果的判定。因此,有必要寻找新的候选基因,并对其进行克隆、表达、纯化,从而进行免疫学特性及相关功能的初步鉴定,为包虫病的防治奠定基础。硫氧还蛋白过氧化物酶(Thioredoxin peroxidase,TPx)属于过氧化物酶超家族(Prx)中的一员,广泛存在于生物体中,它以硫氧还蛋白作为电子供体去除机体内的活性氧类物质,不仅具有强大的抗氧化功能,而且在细胞分化、凋亡和细胞增殖等方面都具有调节作用。目前,有关线虫和吸虫抗氧化物酶方面的研究已很多,但是绦虫抗氧化酶的研究还较少。1998年,Salinas等研究发现:在体外培养条件下,细粒棘球绦虫的幼虫原头蚴(PSC)可以代谢H2O2,而在其体内检测不到过氧化氢酶和谷胱甘肽过氧化物酶的活性,因此推测EgTPx在保护机体免受氧化损伤过程中起着关键的作用,其对于棘球蚴在人和其它中间宿主内脏器官中存活是十分必要的,因而成为寄生虫免疫研究的重点。本研究根据细粒棘球绦虫硫氧还蛋白过氧化物酶(EgTPx)的基因序列[AF478688]设计引物,扩增EgTPx基因,分别构建原核和真核重组质粒,利用大肠杆菌和毕赤酵母两种表达系统分别对其进行表达和鉴定,以期为包虫病分子疫苗的研制提供新的候选基因。研究内容主要包括:细粒棘球绦虫硫氧还蛋白过氧化物酶基因的克隆、原核表达及免疫学特性鉴定;细粒棘球绦虫硫氧还蛋白过氧化物酶(EgTPx)基因在毕赤酵母中的分泌表达及活性鉴定。首先,以细粒棘球绦虫原头蚴的RNA为模板,根据互联网GenBank检索出的细粒棘球绦虫硫氧还蛋白过氧化物酶基因序列设计引物,采用RT-PCR技术扩增目的基因,将目的基因克隆到原核表达载体pET-41b ,转化入大肠杆菌BL21,对重组基因进行DNA测序,利用DNAman、NCBI/BLAST公共数据库对目的基因的同源性进行比较分析正确后进行原核诱导表达,并经亲和层析法纯化重组蛋白(rEgTPx)——EgTPx/GST。结果显示:PCR扩增得到582bp的目的基因,该基因与互联网检索的基因序列同源性为99.83%,氨基酸序列同源性为100%,表达并纯化出rEgTPx,分子量约54KDa。由此,大量制备了重组蛋白rEgTPx,为后续实验提供了抗原物质。其次,用纯化的重组蛋白rEgTPx作为抗原免疫小鼠制备抗血清,通过ELISA及Western blot对重组蛋白rEgTPx的免疫学特性进行初步研究。间接ELISA检测结果表明制备的抗血清能与重组蛋白rEgTPx发生特异性免疫识别,免疫过程中抗体滴度呈现连续动态上升的趋势,其抗血清的效价达到1:25 6000。Western blot结果显示:获得的抗血清能与rEgTPx和天然原头蚴抗原发生特异性反应,表明重组蛋白rEgTPx具有原头蚴天然TPx蛋白的免疫活性,也间接说明rEgTPx可能很好地保持了原头蚴天然TPx的生物学功能表位,有望成为包虫病高效疫苗的候选靶抗原,并为EgTPx免疫诊断提供有效的抗原物质。另外,原核表达系统也能表达出具有活性的EgTPx蛋白,但该类系统缺乏必需的蛋白质翻译后修饰及折叠机制,进而可能影响真核来源的重组产物的生物学活性。毕赤酵母(Pichia pastoris)表达系统以其无可比拟的优越性成为近年来发展迅速、应用广泛的真核表达系统。到目前为止,超过500种外源蛋白的基因被克隆至毕赤酵母表达系统中并被成功地表达。因此,本研究利用毕赤酵母表达系统表达EgTPx,以期获得更具天然活性的EgTPx蛋白。首先,从棘球蚴包囊中分离出原头蚴,抽提总RNA,采用RT-PCR的方法,获取EgTPx的cDNA片段并克隆到分泌型表达载体pPIC9K信号序列α因子之后,构建重组表达质pPIC9K-EgTPx,用限制性内切酶Bgl II将其线性化后经电转化法转化毕赤酵母GSll5,在MD平板上筛选His+克隆,采用梯度浓度G418抗性筛选高拷贝转化子,提取酵母基因组DNA进行PCR鉴定及Mut+表型鉴定;在AOX1启动子调控下,阳性克隆经摇瓶发酵和1%甲醇诱导后对表达产物进行SDS-PAGE检测和western blot鉴定,结果表明目的蛋白分子量大约为22KDa,具有良好的免疫原性。不同浓度的H202检测其抗氧化活性的实验表明,表达EgTPx的酵母细胞对H202的耐受能力明显高于对照组。本研究在毕赤酵母表达系统中实现了EgTPx的分泌表达,为深入探讨EgTPx的生物学功能研究奠定物质基础。更多还原

【Abstract】 Echinococcosis is a cosmopolitan zoonosis caused by adult or larval stages of cestodes belonging to the genus Echinococcus, which is badly harm for human health and stockbreeding production. Now the therapy of Cystic Echinococcosis(CE) in humans is mainly according to surgery or drug treatment,but the surgical operation has a large of damnification for human body and can not ensure that they will not infection again. Meanwhile, patients with CE who take drug treatment may bring fearful ill reaction. So it is impossible to cure CE completely. Larval infection (hydatid disease, hydatidosis) is characterized by long-term growth of metacestode (hydatid) cysts in the intermediate host and easy to diffuse, we have no effective prevent measure as yet. so the early diagnosis and prevent for most human cases of CE is become more and more important. Because it is difficult to diagnose CE with traditional etiology method, often use physical imaging methods and immunodiagnosis as assistant measure.The immunodiagnosis with its low expense, simple operation and high specificity become important measure in CE diagnosis. But there is a lack of standard and specific antigen which interfere the experiment sensitivity and result determination. Therefore, finding new candidate gene of E. granulosus, their expression, purification, and identification of their immunological characters is necessary .The TPx family is a large family of antioxidant proteins universally found in all living organisms, from prokaryotes to eukaryotes. TPx functions as an antioxidant to remove the reactive oxygen species (ROS) and H2O2 derived from normal cellular metabolism using thioredoxin as the electron donor. TPx have also been implicated in oxidative signalling mechanisms regulating apoptosis, cell differentiationand cell proliferation. In parasites, antioxidant enzymes have been extensively characterized in parasitic nematodes and trematodes, where they play a key role in protecting these organisms from the potentially damaging effects of ROS and host-activated leukocytes, but few studies have been carried out on cestodes. It has been demonstrated that protoscoleces(PSC)of Echinococcus granulosus(hydatid cysts)can metabolize hydrogen peroxide in vitro, but, given that neither catalase nor glutathione peroxidase activities were detectable, EgTPx may play a role in protecting the parasite from oxidative damage. so the TPx was considered to be the hotspot in present research.In the present study, thioredoxin peroxidase(TPx) gene from Echinococcus granulosu(sEg) was cloned by RT-PCR, the prokaryotic and eukaryotic recombinant plasmid was constructed and expressed. Then the immunological and antioxidant characters of the recombinant thioredoxin peroxidase in E. coli and Pichia pastoris expression system was identified respectively. Providing valuable candidate gene to develop vaccine for preventing Eg.Firstly, total RNA was extracted from PSC of cysts of sheep. The specific primers were designed according to published nucleotide sequence of NCBI/GenBank database. The TPx gene of Eg was amplified by RT-PCR and insert into pET-41b vector and transfer into E.coil BL21 to construct the expressed recombinant plasmid pET-41b-EgTPx. Then sending the recombinant plasmid pET-41b-EgTPx to company for sequencing. The homology of EgTPx gene was analyzed using DNAman software and NCBI/BLAST database. Then the genetically engineered bacteria pET-41b- EgTPx were induced by IPTG, the expression products were analyzed by SDS- PAGE and then the recombinant protein EgTPx/GST(rEgTPx) was purified using Glutathione Sepharose 4B affinity column. As a result, a gene sequence of 582 bp has been successfully amplified by RT-PCR .Comparing the DNA and deduced amino acid sequence with the published EgTPx gene sequence of Eg to revealed 99.83%and 100% homology respectively. The SDS- PAGE analysis showed that the EgTPx was expressed in E. col.i and the relative molecular weight (Mr) of expressed fusion protein was 54 000. Secondly, the purified rEgTPx was used as antigen to immunize mice and obtain the purified serum for testing the biological activity of rEgTPx by western blot and ELISA. Western blotting analysis show that the antiserum was specific against the rEgTPx protein and PSC antigen, and the titer of the antiserum detected by ELISA was 1 :25 6000 .Thirdly, prokaryotic expression systerm is lack of correctly fold the foreign protein and perform other post-translational modifications mechanism, so E. coli expressed proteins also tend to retain their amino-terminal methionine, which may affect protein stability. The methylotrophic yeast Pichia pastoris, as an eukaryotic expression system, has been a favorite system for expressing heterologous proteins due to its unexampled advantages. In this paper, we attempted to produce EgTPx with biologically activity using P. pastoris expression system . A cDNA fragment from PSC was cloned by RT-PCR and inserted into expression vector pPIC9K, designated as pPIC9K-EgTPx. The recombinant vector was linearized by restriction enzyme Bgl II and transformed into GS115 by electroporation.Three recombinant strains containing multiple copies were obtained by G418 resistance screening and then they were identified by PCR analysis. Under the control of the promoter AOX1, the gene was induced with 1% methanol to secret EgTPx into culture medium. The culture supernatant was analyzed by SDS-PAGE and Western blot, which showed that the protein of EgTPx has a molecular mass of 22 KDa and could bind to EgTPx specific polyclonal antibodies. An in vivo assay showed that the secreted EgTPx could significantly increased the tolerance and survival of the cells existing in high concentrations of H2O2 comparing with controls. The successful cloning and expression of EgTPx in Pichia pastoris has laid a foundation for its further application.更多还原