弓形虫GRA1基因核酸疫苗的免疫研究

发布时间：2018-02-09 12:10

本文关键词： 刚地弓形虫 GRA1基因核酸疫　出处：《吉林大学》2010年硕士论文　论文类型：学位论文

【摘要】： 弓形虫是环境中有致畸效应的病原群(风疹病毒、疱疹病毒及弓形虫等)之一,受染的母体可经胎盘血将其传给胎儿,引起先天性弓形虫病,表现为胎儿发育异常,如脑积水、流产、早产、眼底损害及智力低下。据有关资料报道,5%的胎儿畸形及24%的流产、死胎系由弓形虫引起。我国已有18个省市自治区的调查研究结果表明人群感染率为5.17%,受感染的孕妇先天性畸形儿的发生率为8%,与未感染者相比明显增高。此外,恶性肿瘤患者长期使用免疫抑制剂或免疫功能受损、器官移植患者等,出现全身性泛发性弓形虫病的报告在逐渐增加。目前尚未找到理想的防治该病的药物,且由于寄生虫耐药性的产生以及药物残留等问题的存在,使弓形虫疫苗的研究成为新的趋势。为了验证GRA1基因疫苗对弓形虫感染的免疫保护作用。本课题以弓形虫总RNA为模板,利用设计合成的一对引物,通过方法体外扩增RT-PCR基因GRA1片段,用DNA重组cDNA技术将弓形虫基因与真核表达载体GRA1相连,构建pVAXl重pVAX1-GRA1组真核表达质粒,并将其转染细胞,验证其在真核细胞中的表达；将Hela重组真核表达质粒注射免疫小鼠研究其免疫保护性。结果表明,pVAX1-GRA1扩增出573bp的开放阅读框,经GRA1比对显示同源性为98%；用间接免Blast疫荧光方法在重组质粒转染后的细胞中检测到特异蛋白；通过Hela试验证实所得蛋白具有一定的反应原性；核酸疫苗免疫保护结果表明,随Western blot着免疫次数的增加抗体滴度逐渐增加,试验组与对照组相比差异显著(P＜0.01),CD4+与CD8+T淋巴细胞数与对照组相比差异显著(P＜0.01)。弓形虫RH速殖子攻击感染小鼠后,小鼠存活时延长,与PBS对照组及对照组差异显著pVAXl(P＜0.05)。
[Abstract]:Toxoplasma gondii is teratogenic effect in the environment of pathogens (rubella virus, herpes simplex virus and Toxoplasma gondii), maternal infected by the placental blood to the fetus, caused by congenital toxoplasmosis, manifestations of fetal abnormalities such as hydrocephalus, abortion, premature birth, fundus damage and mental retardation. According to the information reported, 5% fetal malformation and 24% miscarriages, stillbirths caused by Toxoplasma gondii. Research in China has 18 provinces and autonomous regions of the results showed that the infection rate of 5.17%, pregnant women with congenital malformation of the infected rate was 8%, compared with uninfected patients significantly increased. In addition, malignant tumor of the long-term use of immunosuppressive agents or impaired immune function, organ transplant patients, systemic disseminated toxoplasmosis report increased gradually.
Has yet to find an ideal drug for the prevention of the disease, and because the parasite drug resistance and drug residues and other problems, the study of Toxoplasma gondii vaccine has become a new trend. In order to verify GRA1 gene vaccine on the immune protective effect of Toxoplasma gondii infection. The arch insect total RNA as template, using a design for primer synthesis, RT-PCR gene fragment by GRA1 method in vitro, using DNA technique of recombinant cDNA gene of Toxoplasma gondii and eukaryotic expression vector GRA1 to construct pVAXl pVAX1-GRA1 group with eukaryotic expression plasmid, and transfected cells, to verify its expression in eukaryotic cells; Hela recombinant eukaryotic expression research the immune protection in mice immunized with plasmid injection. The results showed that pVAX1-GRA1 amplified the open reading frame of 573bp by GRA1 comparison showed 98% homology with Blast; indirect immunofluorescence method in free recombinant plasmid To detect specific protein transfected cells; by Hela test confirmed that the protein has certain immunogenicity; nucleic acid vaccine immune protection. The results showed that Western blot increased gradually with the increase of the number of immune antibody titer test, compared with the control group (P < 0.01), CD4+ and CD8+T lymphocyte count and control group compared with significant difference (P < 0.01). RH of Toxoplasma gondii tachyzoite infected mice, the mice survived when prolonged significantly in pVAXl group and control group differences and PBS (P < 0.05).

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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