本文關鍵詞： 剛地弓形蟲 GRA1基因 核酸疫　出處：《吉林大學》2010年碩士論文　論文類型：學位論文

【摘要】： 弓形蟲是環(huán)境中有致畸效應的病原群(風疹病毒、皰疹病毒及弓形蟲等)之一,受染的母體可經胎盤血將其傳給胎兒,引起先天性弓形蟲病,表現(xiàn)為胎兒發(fā)育異常,如腦積水、流產、早產、眼底損害及智力低下。據(jù)有關資料報道,5%的胎兒畸形及24%的流產、死胎系由弓形蟲引起。我國已有18個省市自治區(qū)的調查研究結果表明人群感染率為5.17%,受感染的孕婦先天性畸形兒的發(fā)生率為8%,與未感染者相比明顯增高。此外,惡性腫瘤患者長期使用免疫抑制劑或免疫功能受損、器官移植患者等,出現(xiàn)全身性泛發(fā)性弓形蟲病的報告在逐漸增加。 目前尚未找到理想的防治該病的藥物,且由于寄生蟲耐藥性的產生以及藥物殘留等問題的存在,使弓形蟲疫苗的研究成為新的趨勢。為了驗證GRA1基因疫苗對弓形蟲感染的免疫保護作用。本課題以弓形蟲總RNA為模板,利用設計合成的一對引物,通過方法體外擴增RT-PCR基因GRA1片段,用DNA重組cDNA技術將弓形蟲基因與真核表達載體GRA1相連,構建pVAXl重pVAX1-GRA1組真核表達質粒,并將其轉染細胞,驗證其在真核細胞中的表達；將Hela重組真核表達質粒注射免疫小鼠研究其免疫保護性。結果表明,pVAX1-GRA1擴增出573bp的開放閱讀框,經GRA1比對顯示同源性為98%；用間接免Blast疫熒光方法在重組質粒轉染后的細胞中檢測到特異蛋白；通過Hela試驗證實所得蛋白具有一定的反應原性；核酸疫苗免疫保護結果表明,隨Western blot著免疫次數(shù)的增加抗體滴度逐漸增加,試驗組與對照組相比差異顯著(P＜0.01),CD4+與CD8+T淋巴細胞數(shù)與對照組相比差異顯著(P＜0.01)。弓形蟲RH速殖子攻擊感染小鼠后,小鼠存活時延長,與PBS對照組及對照組差異顯著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).

【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

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本文編號：1497895