【作者】 吴翔；

【导师】 汪世平； 舒衡平；

【作者基本信息】 中南大学 ， 病原生物学， 2007， 博士

【摘要】 研究背景:弓形虫是一种专性细胞内寄生的机会致病原虫,生活史复杂,涉及的组织器官多,可引起人兽共患的弓形虫病。其危害严重程度在再现疾病之中仅次于结核;孕妇感染弓形虫可引起流产、早产、畸胎、死胎,婴幼儿先天性弓形虫病,小儿智力障碍等,对人口素质和优生优育和计划生育有严重影响。近年来,弓形虫脑炎已成为艾滋病患者的主要死亡原因之一;也是器官移植患者死亡和精神病发病的主要原因之一。危害如此严重,诊断、治疗和预防却还存在很多问题亟需解决。治疗尚无十分理想的药物,采用的化学药物有治疗周期长,药物毒副作用大,不能根治等缺陷,加上至今还无一种药物能够杀灭包囊内的虫体,加之弓形虫的重复感染十分迅速,故虽经多方面的努力,弓形虫病仍未得以很好地遏制。因此,研制有效的新药和安全高效的弓形虫病疫苗,已成为人们的迫切要求,而廉价、安全、高效的疫苗无疑是最为经济、实用的防治措施。弓形虫病疫苗候选分子的筛选一直是研究中的瓶颈,也是研究的焦点和热点。本工作在前期研究的基础上进一步筛选、鉴定更有效的疫苗候选基因或药物分子靶标,制备核酸疫苗进行动物实验并阐明这个基因的功能、作用机制及其潜在的应用价值。研究目的:（1）通过制备具有保护性的单克隆抗体,筛选、鉴定弓形虫病疫苗的新的候选基因。（2）构建该基因的DNA疫苗,观察其动物保护效果以及对宿主机体免疫系统的影响。（3）应用RNA干扰技术,使获得的候选基因在转录后水平沉默,从而使其所表达的蛋白下降或者缺失,以期进一步阐明该基因的功能和作用机制,对该基因的应用前景进行评估,并希望得到弓形虫的减毒虫株,用于制备减毒活疫苗。研究方法:（1）应用杂交瘤技术获得抗弓形虫的单克隆抗体,体内外实验观察其保护性效果。以弓形虫单克隆抗体作为探针免疫筛选弓形虫速殖子cDNA文库,获得其对应的基因序列。经Blast(http://www.ncbi.nlm.nih.gov/BLAST和（http://www.toxodb.org/ToxoDB.html.）进行同源性比较,登录GenBank并获得新基因登录序列号。（2）采用双色免疫荧光抗体定位和真核细胞内表达等方法对该基因编码的蛋白质进行鉴定,应用生物信息学对基因编码蛋白的B细胞表位、氨基酸序列、蛋白质结构等进行预测和同源性分析。（3）以携带wx2基因的pBluescript7C3-C3质粒DNA为模板,通过PCR扩增基因wx2的ORF;以携带wx基因的pBluescript2B9-G1质粒DNA为模板,经PCR扩增获得wx基因的ORF,构建wx2和wx新基因的DNA疫苗并免疫昆明小鼠,观察小鼠死亡时间和检测实验动物的淋巴细胞转化率、脾细胞CD₄^*与CD₈⁺淋巴细胞比值、IFN-γ、血清特异抗体等,以期阐明对机体免疫系统的影响。（4）应用RNA干扰技术构建新基因wx2,wx逆病毒表达载体,与真核表达质粒pcDNA3-wx2,pcDNA3-wx一起,经脂质体共转染真核细胞观察体外表达效果;经电穿孔法导入弓形虫体内,获得体内能持续表达siRNA分子的可遗传的RNA干扰虫株。采用Western-blotting,RT-PCR,Northern-blotting等方法鉴定其蛋白质及mRNA水平变化效果;并对有基因沉默效果的干扰虫株进行动物感染实验,观察其的毒力变化;以研究减毒活疫苗的潜在价值。研究结果:（1）获得了两株抗弓形虫的单克隆抗体7C3-C3,289-G1,体外保护性实验提示能抑制弓形虫对宿主细胞的侵袭以及在宿主细胞内的繁殖,其细胞感染率分别为28%和32%,对照组为85.2%;50个HeLa细胞中纳虫泡内平均虫体数目为5.18±3.34与5.50±2.36,对照组为11.12±4.29。被动转移实验提示其能够延长RH株弓形虫攻击小鼠的生存时间（7.2±0.42和7.0±1.41d,对照组为5.0d）,表明这两个单抗具有一定的抗弓形虫感染的保护力。（2）通过免疫筛选弓形虫速殖子cDNA文库,获得了两个单抗所对应的基因序列,经同源性比对发现它们为新基因;GenBank的登录号为AY238892和AY208994。通过双色免疫荧光定位和体外表达以及生物信息学分析对两个新基因进行了鉴定,证明WX2为一新的功能膜分子,分子量为49kDa;WX为弓形虫60S核糖体L7蛋白,分子量为47kDa,位于胞质中。（3）成功构建了新基因wx2,wx的真核表达质粒,作为DNA疫苗免疫动物显示pcDNA3-wx2与pcDNA3-wx能显著延长弓形虫RH株攻击感染的生存时间,分别达289.14±46.81h和284.29±47.30h,对照组仅存活176.4±1.98h和172±1.88h,而且攻击感染30天后疫苗接种组21只实验鼠中均有4只小鼠存活,显示其潜在的应用价值。对免疫鼠研究发现,pcDNA3-wx2能刺激机体产生较高水平的IFN-γ（P＜0.05）;pcDNA3-wx2与pcDNA3-wx都能使宿主脾细胞CD₄⁺/CD₈⁺比值下降,及产生特异性抗体,与对照组相比,P值＜0.05;但抗体效价的高低与保护力的强弱并不平行;提示该DNA疫苗主要以CD₈⁺的CTL细胞途径激发机体的抗弓形虫效应。（4）成功构建了5个针对基因wx2,wx的干扰表达质粒,获得了2株部分沉默的RNA干扰虫株wx2b-i和wxB-i虫株。经过体外表达以及RT-PCR,Northern-blotting等鉴定,证实基因wx2,wx mRNA水平及蛋白质的表达均有部分下降。干扰虫株wx2b-i腹腔接种小鼠后其存活时间明显延长,平均存活时间为235.4±15.4h,阴性组C-i和野生型RH株组分别为161.2±11.98h与165.4±6.09h,统计学分析具有显著性差异,发病及死亡时间推迟,表明干扰株的毒力有所降低。结论:（1）抗弓形虫的单克隆抗体7C3-C3,289-G1对于弓形虫的感染具有一定的保护性,能抑制弓形虫对宿主细胞的侵袭以及在宿主细胞内的繁殖;被动转移实验提示能够延长RH株弓形虫攻击小鼠的生存时间。（2）WX2为一新的弓形虫功能膜分子,分子量为49kDa;WX为弓形虫60S核糖体L7蛋白,分子量为47kDa,定位于胞质中。wx2,wx是两个较好的弓形虫疫苗候选分子。（3）成功建立了含新基因wx2,wx的DNA疫苗,能显著延长弓形虫强毒（RH株）攻击感染动物的生存时间,显示其潜在应用价值。对宿主免疫系统的影响观察提示其主要以CD₈⁺的CTL细胞途径激发机体的抗弓形虫效应。（4）成功构建了5个针对基因wx2,wx的干扰表达载体,并获得了两株部分沉默的RNA干扰虫株wx2b-i、wxB-i,其中wx2b-i虫株的毒力有所降低,为进一步研究基因wx2,wx的功能与作用机制奠定了基础。更多还原

【Abstract】 Background:Toxoplasma gondii is an obligate intracellular protozoan parasite which could infect nearly all kinds of animals and birds and involve multiple host organs.The infection can result in toxoplasmosis.In humanbeings,severe disease in adults exists mainly in mmunosuppressed patients.Toxoplasmosis was regarded as an important recurring disease only second to tuberculosis which was seriously harmful to human heath.The infected pregnant woman could transmit the parasite to fetus through placenta,leading to abortion,stillbirth,neonatal deformety, mental symptoms and congenital toxoplasmosis of survival infants.It brought very serious effects on the eugenesis and family planning in our country.Recently,it was reported that Toxoplasma encephalitis is the most important manifestation and the main cause of death of in immunodeficiency individuals,such as AIDS patients et al.Furthermore, toxoplasmosis is also the main cause of death in the patients who received organ transplantation and is one of the main reasons of first-episode in schizophrenia and cryptogenic epilepsy patients.At present,there are still difficulties in prevention,diagnosis and treatment of the disease. Chemotherapy is not satisfactory because of the toxicity,need for long-term treatment and rapid re-infection by the parasite;moreover, there is no drug that can kill the encysted bradyzoites.As a result, toxoplasmosis has not been well controlled in the world.The development of cheap,safe and effective vaccines and new therapeutic drugs become priorities in the control of the disease.Therefore,the screening of good candidate molecules for the toxoplasmosis vaccine is not only a research bottleneck,but also a focus and hot point in the currently vaccine research.On the basis of the previous studies,we devoted ourselves to find and identify more effective vaccine candidate genes or drug target molecules,and then to produce DNA vaccines, illuminate the function and mechanism of the gene,as well as its potential value.Objective:（A）Through preparing new monoclonal antibodies that possessing protection effect against T.gondii,to find and identify new candidate genes for the vaccine of toxoplasmosis.（B）Constructing the DNA vaccines corresponding to the genes screened before,and surveying their protection effect in animals and the mechanism of the effect.（C）RNAi technique was used to silence the genes at the post-transcription level to make its expressed protein being loss or declined,so as to illuminate the function of the genes and evaluate the practical prospects of the genes and their DNA vaccine.Methods:（1）Monoclonal antibodies against Toxoplasma gondii were produced by hybridoma technology,and then their protective effect in vitro and in vivo experiments were evaluated.Using these monoclonal antibodies as probes to immunoscreen toxoplasmosis tachyzoite cDNA library,the corresponding gene sequences were obtained.Afterward the homology analysis was blast at websites:http://www.ncbi.nlm.nih.Gov /BLAST and（http://www.toxodb.Org/ToxoDB.html.）Login GenBank and accessible sequence were acquired.（2）The encoded proteins were identified by immurlofluorescence microcopy and the expressions in eukaryotic cells.The B cell epitope, amino acid sequence and the structure of the proteins encoded by the genes were predicted and homology analysis（including Phylograrn tree and the homology blast）was performed by bioinformation methods.（3）Using plasmid pBluescript 7C3-C3 in which wx2 gene was carried as the template in PCR to amplify the ORF of wx2,and plasmid pBluescript 2B9-G1 in which gene wx was carried as the template in PCR to amplify the ORF of wx,the DNA vaccines with eukaryotic expression vector of the two new genes were constructed.Protective experiments in animals were done and the survival time of challenged mice was observed.Lymphocyte transformation rate,CD₄⁺ and CD₈⁺ lymphocyte ratio of the spleen cells,IFN-γlevel and specific antibody in serum of the immunized animals were examined.（4）RNAi expressed vectors with the new gene wx2 and wx as their targets were constructed.The effect of interference was observed by lipid-mediated transfection of eukaryotic cells in vitro.Plasmid was transfected into the toxoplasma cells by electroporation,so that the silent strains of toxoplasma in which siRNA molecules could be expressed continuously were produced.By Western-blotting,RT-PCR,Northern blotting et al,the interference effect was characterized,and these artificially produced toxoplasma can be used for further study.Result（1）Two monoclonal antibodies against Toxoplasma gondii 7C3-C3, 2B9-G1 were obtained.The protective tests in vitro showed that these proteins could inhibit the invasion and proliferation of T.gondii RH strain in HeLa cells.The cell infection rate was 28%and 32%respectively,and that of the control was 85.2%.The average number of T.gondii tachyzoite of in 50 HeLa cells was 5.18±3.34 and 5.50±2.36,while that of the control was 11.12±4.29.The passive transfer test indicated that these proteins significantly prolonged the survival time of the challenged mice（7.2±0.42days and 7.0±1.41days,while that of the control was 5.0 days）.It was also shown that the two McAbs had certain protection ability against T.gondii infection.（2）Through immunoscreening toxoplasma tachyzoite cDNA library, the gene sequences corresponding to these two monoclonal antibodies were obtained.The accessible numbers were AY238892 and AY208994 in GenBank.By immunofluorescence localization test,expression in vitro and bioinformatics analysis,it was demonstrated that WX2 was a new functional molecule on the membrane of T.gondii of which molecular weight was 49kDa,and WX was a 60S ribosomal L7 protein of T.gondii, a molecule in cytoplasm of which the molecular weight was 47kDa.（3）The DNA vaccines of new genes wx2 and wx were constructed successfully.Protective experiments of DNA vaccines pcDNA3-wx2 and pcDNA3-wx in animals showed that the vaccines could significantly prolong the survival time of the mice challenged by virulent RH strains of Toxoplasma.Comparing with other toxoplasma vaccines against the virulent strains reported currently,the survival time is the longest.The average survival time was 289.14±46.81hours and 284.29±47.30hours respectively.Moreover,30 days after challenge,there were still four mice survived in each group.The study on the impact to the immune system indicated that,pcDNA3-wx2 vaccine could stimulate the animal to produce high level IFN-γ（149.8±24.53ng）,both pcDNA3-wx and pcDNA3-wx2 could induce the decrease of the CD4⁺/CD8⁺ ratio and the production of the specific antibody in the experimental mice.However, the level of antibody titers did not parallel with the degree of protection. It indicated that these two DNA vaccines could stimulate the host resisting Toxoplasma mainly by the way of CD8⁺CTL cells.（4）Five interference expression vectors against Gene wx2,wx were constructed,and two partially silent strains T.gondii wx2b-i and wxB-i, were obtained.At the same time the virulence of wx2b-i partially reduced. It was confirmed that the mRNA and protein expression of wx2 and wx genes were partly reduced which were identified by the in vitro expression,RT-PCR and Northern blotting.Conclusion:（1）Monoclonal antibodies 7C3-C3 and 2B9-G1 have some protective effect against Toxoplasma gondii.They can inhibit the invasion of the parasite to host cells and the propagation of it in the host cell.The passive transfer experiments suggested that they could prolong survival time of the mice challenged by RH strain Toxoplasma gondii.（2）WX2 is a new functional membrane molecule,with 49kDa molecular weight.WX is toxoplasmoa 60S ribosomal L7 protein,with 47kDa molecular weight in cytoplasm.So the corresponding genes wx2 and wx are good new candidates for DNA vaccine of T.gondii.（3）The DNA vaccines of new genes wx2 and wx were constructed successfully.Protective experiments in animals showed that the vaccine could significantly prolong the survival time of the mice challenged with virulent RH strains of Toxoplasma,The time is longer than that acquired by the other DNA vaccines on current reports,which indicated their potential value.The immue mechanism is mainly by the way of CD8⁺ CTL cell.（4）Five interference expression vectors against Gene wx2,wx were constructed,and two partial silent strains wx2b-iand wxB-i were obtained. All these formed a foundation for further study on the function and mechanism of gene wx2,wx.更多还原