【作者】 李曼；

【导师】 沈继龙；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2016， 硕士

【摘要】 背景：弓形虫(Toxoplasma gondii)是一种专性细胞内寄生的原虫,可寄生于人和许多动物的有核细胞内,引起机会性人兽共患性弓形虫病,大多数感染者无临床症状,但对于具有免疫缺陷的人则可引起严重的临床症状甚至导致患者死亡。弓形虫病至今尚无有效的防治药物,疫苗防治是发展方向。但由于弓形虫有着复杂的生活史以及多种入侵宿主途径,虫体具有多种高度多态性的特异性抗原并能逃避宿主免疫系统的攻击,从而导致弓形虫疫苗的研制与其它细菌和病毒性传染病疫苗相比更加复杂。目前对弓形虫疫苗的研究越来越深入,随着分子生物学研究和基因工程的不断发展,使弓形虫疫苗的研发有了很大突破。有研究报告,GRA15疫苗用于昆明小鼠的免疫可产生抗弓形虫感染的保护性免疫,基于文献报道GRA15的疫苗的免疫原性效果,本研究将制备的WH3-GRA15-GFP核酸疫苗直接肌注免疫小鼠,通过观察和检测弓形虫WH3-GRA15-GFP核酸疫苗的免疫原性和免疫保护性,筛选WH3-GRA15-GFP核酸疫苗能否作为抗击弓形虫感染的候选疫苗,以期用于弓形虫病的预防和治疗。目的：利用GRA15基因真核表达重组质粒免疫BALB/c小鼠,检测小鼠体液免疫和细胞免疫,观察WH3-GRA15-GFP核酸疫苗对BALB/c小鼠的免疫保护作用。方法：克隆Chinese 1型弓形虫Wh3株GRA15编码序列将其插入真核表达载体pEGFP-C2中构建重组质粒WH3-GRA15-GFP,脂质体法转染T293细胞并观察体外表达,Western blotting分析鉴定。54只BALB/c小鼠随机分为PBS对照组、空质粒对照组、实验组,每组18只,均通过股四头肌注射方式免疫小鼠,其中实验组每鼠注射WH3-GRA15-GFP重组质粒1001μg,空质粒对照组注射pEGFP-C2质粒1001μg,PBS对照组注射无菌PBS缓冲液100μL。共免疫3次,每次间隔2周。分别于每次注射前和末次注射后第2周检测小鼠血清特异性IgG、IgGl、IgG2a抗体和IL-2、IL-4、IL-10、IFN-γ等细胞因子。末次注射后2周,各组随机挑选13只小鼠腹腔攻击感染弱毒株弓形虫速殖子1000个/只,观察其存活时间,评价免疫效果,5只实验组小鼠检测重组质粒在免疫小鼠体内表达情况。结果：WH3-GRA15-GFP核酸疫苗小鼠体内有表达但不能诱发小鼠产生细胞免疫和体液免疫反应,实验组小鼠血清特异IgG、IgG1、IgG2a随着免疫次数的增加没有显著变化(p>0.05),IL-2、IL-4、IL-10、IFN-y和空白对照组小鼠相比均没有增加(p>0.05)。各组小鼠遭弓形虫攻击感染后的平均存活时间无统计学差异(p>0.05)。结论：WH3-GRA15-GFP核酸疫苗在BALB/c小鼠体内有表达但对鼠弓形虫感染无免疫保护性,可能不宜作为弓形虫有效的疫苗候选分子。更多还原

【Abstract】 Background:Toxoplasma gondii is a specially designed cell in parasitic protozoa and parasitic in the human and many animals in eukaryotic cell, causing zoonotic toxoplasmosis. Most infected people have no clinical symptoms and signs, but in those with the human immunodeficiency virus it can cause serious consequences. There is no effective drug for prevention and treatment of toxoplasmosis, thus vaccine prevention is the direction of development. At present, Toxoplasma gondii vaccine study has not given rise to a practical and effective candidate miolecule which can be used. The reason for the unsuccessfulness may be the complex life cycle of the parasite, with specific antigens of diversification, high degree of variability, multiple invasion pathways, and evasion of the host immune attack mechanism. With the development of molecular biology and gene engineering, a progress has been made in the research of T.gondii vaccine development. Here we used WH3-GRA15-GFP nucleic acid vaccine by intramuscular immunization of mice and detected the immunogenicity and efficacy of immune protection so as to screen the candidate vaccines against Toxoplasma infection.Objective:To construct the recombinant eukaryotic expression plasmids of Toxoplasma GRA15, and to assess the immune protection in BALB/C mice induced by a WH3-GRA15-GFP nuclei acid vaccination.Methods:T. gondii GRA15 coding gene obtained by PCR amplification was inserted into the eukaryotic expression vector pEGFP-C2 to generate recombinant WH3-GRA15-GFP. T293 cells were transfected by Liposome method in vitro and the fusion protein expression was identified by Western blotting. Fifty-four BALB/C mice were randomly divided into experimental group, empty plasmid group, and control group, with 18 mice in each.The mice were immunized via injection in the quadriceps femoris.The experimental group was injected with 100μg of the recombinant plasmid WH3-GRA15-GFP, the empty plasmid group was injected with 100μg of the pEGFP-C2 plasmid, and the PBS group was injected with 100μL of PBS buffer. Each immunization was done a total of three times with an interval of 2 weeks. Before each injection and two weeks after the final injection, levels of serum IgG, IgG1, and IgG2a antibodies and cytokines(IL-2、IL-4、IL-10、IFN-γ) were measured. Thirteen mice in all of the groups were challenged intraperitoneslly with 1,000 tachyzoites of the strain of T. gondii two weeks after the final injection to observe their survial time and evaluate immunity.Results:WH3-GRA15-GFP nucleic acid was found to be expressed in mice but failed to induce effective cellular and humoral immune response. Compared with control group,both the levels of the specific antibodies (IgG, IgG1, IgG2a) and cytokines (IL-2, IL-4,IL-10, and IFN-γ) and the number of deaths of mice did not reveal a statistically significant change (P>0.05).Conclusions:Dense granular protein of GRA15 was not effectively enough to serve as Toxoplasma vaccine candidate molecule. The GRA15 prediction protein is not stable and has less antigen epitopes coupled with a relatively simple 3D structure.WH3-GRA15-GFP DNA vaccine of T.gondii may not be an effective vaccine candidate molecule due to its failure to induce immune protection in BALB/c mice.更多还原