發(fā)布時(shí)間：2018-02-09 12:10

  本文關(guān)鍵詞： 剛地弓形蟲 GRA1基因 核酸疫　出處：《吉林大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 弓形蟲是環(huán)境中有致畸效應(yīng)的病原群(風(fēng)疹病毒、皰疹病毒及弓形蟲等)之一,受染的母體可經(jīng)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(chǎn)、早產(chǎn)、眼底損害及智力低下。據(jù)有關(guān)資料報(bào)道,5%的胎兒畸形及24%的流產(chǎn)、死胎系由弓形蟲引起。我國已有18個(gè)省市自治區(qū)的調(diào)查研究結(jié)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(bào)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(chǎn)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(yàn)證GRA1基因疫苗對弓形蟲感染的免疫保護(hù)作用。本課題以弓形蟲總RNA為模板,利用設(shè)計(jì)合成的一對引物,通過方法體外擴(kuò)增RT-PCR基因GRA1片段,用DNA重組cDNA技術(shù)將弓形蟲基因與真核表達(dá)載體GRA1相連,構(gòu)建pVAXl重pVAX1-GRA1組真核表達(dá)質(zhì)粒,并將其轉(zhuǎn)染細(xì)胞,驗(yàn)證其在真核細(xì)胞中的表達(dá)；將Hela重組真核表達(dá)質(zhì)粒注射免疫小鼠研究其免疫保護(hù)性。結(jié)果表明,pVAX1-GRA1擴(kuò)增出573bp的開放閱讀框,經(jīng)GRA1比對顯示同源性為98%；用間接免Blast疫熒光方法在重組質(zhì)粒轉(zhuǎn)染后的細(xì)胞中檢測到特異蛋白；通過Hela試驗(yàn)證實(shí)所得蛋白具有一定的反應(yīng)原性；核酸疫苗免疫保護(hù)結(jié)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(yàn)組與對照組相比差異顯著(P＜0.01),CD4+與CD8+T淋巴細(xì)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(shí)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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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本文編號：1497895