具有潜在疫苗价值的弓形虫新基因wx2，wx的筛选与鉴定
发布时间：2018-09-01 17:52
【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CD_4~*与CD_8~+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CD_4~+/CD_8~+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CD_8~+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CD_8~+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。
[Abstract]:Research background:
Toxoplasma gondii is a specific intracellular parasitic opportunistic pathogenic protozoan with a complex life cycle, involving many tissues and organs, and can cause toxoplasmosis zoonosis. The severity of toxoplasmosis is second only to tuberculosis in the recurrence of the disease; Toxoplasma gondii infection in pregnant women can cause abortion, premature delivery, teratoxoplasmosis, stillbirth, congenital toxoplasmosis in infants and children's intelligence. In recent years, Toxoplasma gondii encephalitis has become one of the main causes of death in AIDS patients and one of the main causes of death and psychosis in organ transplant patients. There are no ideal drugs for the treatment of toxoplasmosis. The chemical drugs used have many defects, such as long treatment cycle, toxic side effects, and no drug can kill the worms in the cysts, and the repeated infection of Toxoplasma gondii is very rapid. Therefore, despite many efforts, toxoplasmosis has not been well controlled. The preparation of new effective drugs and safe and efficient vaccine against Toxoplasma gondii has become an urgent requirement of people, and inexpensive, safe and efficient vaccine is undoubtedly the most economical and practical preventive measures. One-step screening, identification of more effective vaccine candidate genes or drug molecular targets, preparation of nucleic acid vaccine for animal experiments and to clarify the function of this gene, the mechanism of action and its potential application value.
Research purposes:
(1) To screen and identify new candidate genes for toxoplasmosis vaccine by preparing protective monoclonal antibodies.
(2) To construct the DNA vaccine of the gene and observe its protective effect on the host immune system.
(3) RNA interference technique was used to silence the candidate gene at the post-transcriptional level so that the expressed protein could be decreased or deleted, so as to further elucidate the function and mechanism of the gene, evaluate the application prospect of the gene, and hope to obtain attenuated Toxoplasma gondii strain for the preparation of live attenuated vaccine.
Research methods:
(1) Monoclonal antibodies against Toxoplasma gondii were obtained by hybridoma technique and their protective effects were observed in vitro and in vivo. The corresponding gene sequences were obtained by screening the DNA Library of Toxoplasma gondii tachyzoites with monoclonal antibodies as probes. Blast (http://www.ncbi.nlm.nih.gov/BLAST) and (http://www.toxodb.org/ToxoDB.html.) Homology comparison, login GenBank and get the new gene accession sequence number.
(2) The protein encoded by the gene was identified by immunofluorescent antibody localization and eukaryotic cell expression. The B cell epitope, amino acid sequence and protein structure were predicted and analyzed by bioinformatics.
(3) The ORF of wx2 gene was amplified by PCR using pBluescript7C3-C3 plasmid DNA carrying wx2 gene as template; the ORF of Wx gene was obtained by PCR using pBluescript2B9-G1 plasmid DNA carrying Wx gene as template; the DNA vaccine of wx2 and Wx gene was constructed and immunized Kunming mice to observe the death time of mice and detect the lymphocyte of experimental animals. The transformation rate, the ratio of CD_4~* to CD_8~+ lymphocyte, IFN-gamma and serum specific antibody were used to elucidate the effect on the immune system.
(4) Using RNA interference technology to construct a new gene wx2, Wx retroviral expression vector, together with the eukaryotic expression plasmid pcDNA3-wx2, pcDNA3-wx, the eukaryotic cells were co-transfected by liposome to observe the effect of expression in vitro; Toxoplasma gondii was transfected by electroporation, and the inheritable RNA interference strain was obtained. Ng, RT-PCR, Northern-blotting and other methods were used to identify the effect of protein and mRNA level changes. Animal infection experiments were carried out to observe the virulence changes of interfering insect strains with gene silencing effect, and to study the potential value of live attenuated vaccine.
Research findings:
(1) Two monoclonal antibodies against Toxoplasma gondii, 7C3-C3,289-G1, were obtained. Protective experiments in vitro showed that the infection rate of the host cells was 28% and 32% respectively, and that of the control group was 85.2%. The average number of parasites in the Nanovesicles of 50 HeLa cells was 5.18 (+ 3.34) and 5.50 (+ 2.36), respectively. The survival time of mice attacked by RH strain of Toxoplasma gondii was prolonged (7.2 (+ 0.42) and 7.0 (+ 1.41) days, while that of control group was 5.0 days), indicating that the two McAbs had certain protective effect against Toxoplasma gondii infection.
(2) Two McAbs were cloned from the DNA Library of Toxoplasma gondii tachyzoites by immunoscreening and identified as new genes by homology comparison. GenBank's login numbers were AY238892 and AY208994. Two new genes were identified by immunofluorescence localization, in vitro expression and bioinformatics analysis. A novel functional membrane molecule with a molecular weight of 49 kDa and a WX ribosomal L7 protein of 60S of Toxoplasma gondii, with a molecular weight of 47 kDa, is located in the cytoplasm.
(3) Eukaryotic expression plasmids of new genes wx2 and Wx were successfully constructed. As DNA Vaccine Immunized animals, pcDNA3-wx2 and pcDNA3-wx significantly prolonged the survival time of RH strain of Toxoplasma gondii infected by attack infection, reaching 289.14, 46.81 h and 284.29, 47.30 h, respectively. The control group only survived 176.4, 1.98 h and 172, 1.88 h, and 21 mice in the vaccination group Four of the mice survived, indicating their potential application value. Studies on immunized mice showed that pcDNA3-wx2 could stimulate the production of higher levels of IFN-gamma (P The level of the DNA vaccine was not parallel to the protective power, suggesting that the anti-Toxoplasma gondii effect of the DNA vaccine was mainly stimulated by CD 8~+ CTL cell pathway.
(4) Five interfering expression plasmids were successfully constructed and two partially silenced RNA interfering strains wx2b-i and wxB-i were obtained. After in vitro expression, RT-PCR, Northern blotting and other identification, the expression of wx2, Wx mRNA and protein were partially decreased. The average survival time was 235.4 + 15.4 H. The virulence of C-i and wild type RH strains in negative group was 161.2 + 11.98 h and 165.4 + 6.09 h respectively. There was significant difference between the two groups. The time of onset and death was delayed, indicating that the virulence of the interfering strains was decreased.
Conclusion:
(1) Anti-Toxoplasma gondii monoclonal antibody 7C3-C3,289-G1 has protective effect on the infection of Toxoplasma gondii, and can inhibit the invasion of Toxoplasma gondii to the host cells and the reproduction of the host cells. Passive transfer experiments suggest that RH strain of Toxoplasma gondii can prolong the survival time of mice.
(2) WX2 is a new functional membrane molecule of Toxoplasma gondii with a molecular weight of 49 kDa, and WX is a 60S ribosomal L7 protein of Toxoplasma gondii with a molecular weight of 47 kDa, which is located in the cytoplasm.
(3) A DNA vaccine containing a novel gene wx2 and Wx was successfully established, which could significantly prolong the survival time of infected animals attacked by Toxoplasma gondii virulent (RH strain) and showed its potential application value.
(4) Five interference vectors targeting wx2 and Wx genes were constructed successfully, and two partially silenced RNA interference strains wx2b-i and wxB-i were obtained. The virulence of wx2b-i strain was decreased, which laid a foundation for further study on the function and mechanism of wx2 and Wx genes.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
本文编号：2217923
[Abstract]:Research background:
Toxoplasma gondii is a specific intracellular parasitic opportunistic pathogenic protozoan with a complex life cycle, involving many tissues and organs, and can cause toxoplasmosis zoonosis. The severity of toxoplasmosis is second only to tuberculosis in the recurrence of the disease; Toxoplasma gondii infection in pregnant women can cause abortion, premature delivery, teratoxoplasmosis, stillbirth, congenital toxoplasmosis in infants and children's intelligence. In recent years, Toxoplasma gondii encephalitis has become one of the main causes of death in AIDS patients and one of the main causes of death and psychosis in organ transplant patients. There are no ideal drugs for the treatment of toxoplasmosis. The chemical drugs used have many defects, such as long treatment cycle, toxic side effects, and no drug can kill the worms in the cysts, and the repeated infection of Toxoplasma gondii is very rapid. Therefore, despite many efforts, toxoplasmosis has not been well controlled. The preparation of new effective drugs and safe and efficient vaccine against Toxoplasma gondii has become an urgent requirement of people, and inexpensive, safe and efficient vaccine is undoubtedly the most economical and practical preventive measures. One-step screening, identification of more effective vaccine candidate genes or drug molecular targets, preparation of nucleic acid vaccine for animal experiments and to clarify the function of this gene, the mechanism of action and its potential application value.
Research purposes:
(1) To screen and identify new candidate genes for toxoplasmosis vaccine by preparing protective monoclonal antibodies.
(2) To construct the DNA vaccine of the gene and observe its protective effect on the host immune system.
(3) RNA interference technique was used to silence the candidate gene at the post-transcriptional level so that the expressed protein could be decreased or deleted, so as to further elucidate the function and mechanism of the gene, evaluate the application prospect of the gene, and hope to obtain attenuated Toxoplasma gondii strain for the preparation of live attenuated vaccine.
Research methods:
(1) Monoclonal antibodies against Toxoplasma gondii were obtained by hybridoma technique and their protective effects were observed in vitro and in vivo. The corresponding gene sequences were obtained by screening the DNA Library of Toxoplasma gondii tachyzoites with monoclonal antibodies as probes. Blast (http://www.ncbi.nlm.nih.gov/BLAST) and (http://www.toxodb.org/ToxoDB.html.) Homology comparison, login GenBank and get the new gene accession sequence number.
(2) The protein encoded by the gene was identified by immunofluorescent antibody localization and eukaryotic cell expression. The B cell epitope, amino acid sequence and protein structure were predicted and analyzed by bioinformatics.
(3) The ORF of wx2 gene was amplified by PCR using pBluescript7C3-C3 plasmid DNA carrying wx2 gene as template; the ORF of Wx gene was obtained by PCR using pBluescript2B9-G1 plasmid DNA carrying Wx gene as template; the DNA vaccine of wx2 and Wx gene was constructed and immunized Kunming mice to observe the death time of mice and detect the lymphocyte of experimental animals. The transformation rate, the ratio of CD_4~* to CD_8~+ lymphocyte, IFN-gamma and serum specific antibody were used to elucidate the effect on the immune system.
(4) Using RNA interference technology to construct a new gene wx2, Wx retroviral expression vector, together with the eukaryotic expression plasmid pcDNA3-wx2, pcDNA3-wx, the eukaryotic cells were co-transfected by liposome to observe the effect of expression in vitro; Toxoplasma gondii was transfected by electroporation, and the inheritable RNA interference strain was obtained. Ng, RT-PCR, Northern-blotting and other methods were used to identify the effect of protein and mRNA level changes. Animal infection experiments were carried out to observe the virulence changes of interfering insect strains with gene silencing effect, and to study the potential value of live attenuated vaccine.
Research findings:
(1) Two monoclonal antibodies against Toxoplasma gondii, 7C3-C3,289-G1, were obtained. Protective experiments in vitro showed that the infection rate of the host cells was 28% and 32% respectively, and that of the control group was 85.2%. The average number of parasites in the Nanovesicles of 50 HeLa cells was 5.18 (+ 3.34) and 5.50 (+ 2.36), respectively. The survival time of mice attacked by RH strain of Toxoplasma gondii was prolonged (7.2 (+ 0.42) and 7.0 (+ 1.41) days, while that of control group was 5.0 days), indicating that the two McAbs had certain protective effect against Toxoplasma gondii infection.
(2) Two McAbs were cloned from the DNA Library of Toxoplasma gondii tachyzoites by immunoscreening and identified as new genes by homology comparison. GenBank's login numbers were AY238892 and AY208994. Two new genes were identified by immunofluorescence localization, in vitro expression and bioinformatics analysis. A novel functional membrane molecule with a molecular weight of 49 kDa and a WX ribosomal L7 protein of 60S of Toxoplasma gondii, with a molecular weight of 47 kDa, is located in the cytoplasm.
(3) Eukaryotic expression plasmids of new genes wx2 and Wx were successfully constructed. As DNA Vaccine Immunized animals, pcDNA3-wx2 and pcDNA3-wx significantly prolonged the survival time of RH strain of Toxoplasma gondii infected by attack infection, reaching 289.14, 46.81 h and 284.29, 47.30 h, respectively. The control group only survived 176.4, 1.98 h and 172, 1.88 h, and 21 mice in the vaccination group Four of the mice survived, indicating their potential application value. Studies on immunized mice showed that pcDNA3-wx2 could stimulate the production of higher levels of IFN-gamma (P The level of the DNA vaccine was not parallel to the protective power, suggesting that the anti-Toxoplasma gondii effect of the DNA vaccine was mainly stimulated by CD 8~+ CTL cell pathway.
(4) Five interfering expression plasmids were successfully constructed and two partially silenced RNA interfering strains wx2b-i and wxB-i were obtained. After in vitro expression, RT-PCR, Northern blotting and other identification, the expression of wx2, Wx mRNA and protein were partially decreased. The average survival time was 235.4 + 15.4 H. The virulence of C-i and wild type RH strains in negative group was 161.2 + 11.98 h and 165.4 + 6.09 h respectively. There was significant difference between the two groups. The time of onset and death was delayed, indicating that the virulence of the interfering strains was decreased.
Conclusion:
(1) Anti-Toxoplasma gondii monoclonal antibody 7C3-C3,289-G1 has protective effect on the infection of Toxoplasma gondii, and can inhibit the invasion of Toxoplasma gondii to the host cells and the reproduction of the host cells. Passive transfer experiments suggest that RH strain of Toxoplasma gondii can prolong the survival time of mice.
(2) WX2 is a new functional membrane molecule of Toxoplasma gondii with a molecular weight of 49 kDa, and WX is a 60S ribosomal L7 protein of Toxoplasma gondii with a molecular weight of 47 kDa, which is located in the cytoplasm.
(3) A DNA vaccine containing a novel gene wx2 and Wx was successfully established, which could significantly prolong the survival time of infected animals attacked by Toxoplasma gondii virulent (RH strain) and showed its potential application value.
(4) Five interference vectors targeting wx2 and Wx genes were constructed successfully, and two partially silenced RNA interference strains wx2b-i and wxB-i were obtained. The virulence of wx2b-i strain was decreased, which laid a foundation for further study on the function and mechanism of wx2 and Wx genes.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
【引证文献】
相关硕士学位论文 前1条
1 邢莹;猪附红细胞体JL-2基因的原核表达及重组蛋白间接ELISA方法的建立[D];延边大学;2012年
，本文编号：2217923
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