發(fā)布時(shí)間：2018-04-10 13:09

  本文選題：弓形蟲 + 肌動(dòng)蛋白解聚因子��； 參考：《吉林大學(xué)》2009年碩士論文

【摘要】： 弓形蟲(Toxoplasma gondii)是一種重要的機(jī)會(huì)性致病原蟲,廣泛寄生于人體及動(dòng)物的組織細(xì)胞內(nèi),引起嚴(yán)重的人獸共患病。據(jù)估計(jì)全世界1/3的人群感染本病,我國人群的感染率為5%～10%。目前尚未找到理想的防治該病的藥物,且由于研制新藥成本費(fèi)用高、藥物殘留以及減毒疫苗毒力返強(qiáng)等問題的存在,使弓形蟲基因工程疫苗的研究成為新的趨勢(shì)。有學(xué)者通過對(duì)弓形蟲抗原及其誘導(dǎo)的免疫反應(yīng)進(jìn)行研究后認(rèn)為有效的疫苗對(duì)該病的防治有一定的作用。為探討ADF基因?qū)蜗x感染的免疫保護(hù)作用,本研究在已知ADF基因序列的基礎(chǔ)上設(shè)計(jì)引物,擴(kuò)增ADF開放閱讀框,將其克隆入原核表達(dá)載體并表達(dá)出重組ADF蛋白,并對(duì)ADF基因在弓形蟲速殖子內(nèi)的分布進(jìn)行定位;同時(shí)用DNA重組技術(shù)將弓形蟲ADF基因與真核表達(dá)載體pVAX1相連,構(gòu)建真核表達(dá)載體,并將重組真核表達(dá)質(zhì)粒轉(zhuǎn)染Hela細(xì)胞,驗(yàn)證其在真核細(xì)胞中的表達(dá);將重組ADF蛋白與真核表達(dá)質(zhì)粒分別免疫小鼠檢測(cè)其免疫保護(hù)力。結(jié)果顯示,擴(kuò)增出365bp的ADF開放閱讀框,經(jīng)Blast比對(duì)顯示同源性為98%,并表達(dá)出17KDa的重組融合蛋白,應(yīng)用免疫熒光抗體技術(shù)將ADF基因定位于速殖子胞質(zhì)內(nèi)及質(zhì)膜上;用間接免疫熒光方法在重組質(zhì)粒轉(zhuǎn)染后的Hela細(xì)胞中檢測(cè)到特異蛋白,通過Western blot試驗(yàn)證實(shí)所得蛋白具有反應(yīng)原性;重組ADF蛋白免疫保護(hù)結(jié)果顯示,隨著免疫次數(shù)的增加抗體滴度逐漸增加,試驗(yàn)組與對(duì)照組相比差異極顯著(P0.01)。CD4+與CD8+T淋巴細(xì)胞數(shù)與對(duì)照組相比差異顯著(P0.01),CD4+/CD8+ T淋巴細(xì)胞數(shù)的值差異不顯著(P0.05)。試驗(yàn)小鼠均未能抵抗弓形蟲強(qiáng)毒株攻蟲感染,但試驗(yàn)組生存時(shí)間延長,與對(duì)照組差異不顯著(P0.05),腦組織中包囊數(shù)減少,減蟲率達(dá)30%;核酸疫苗免疫保護(hù)結(jié)果表明,試驗(yàn)組與對(duì)照組相比,特異性抗體滴度、CD4+與CD8+T淋巴細(xì)胞數(shù)均差異極顯著(P0.01),CD4+/CD8+ T淋巴細(xì)胞數(shù)的值差異不顯著(P0.05)。攻蟲后小鼠生存時(shí)間得到延長,與PBS對(duì)照組差異顯著(P0.05),與pVAX1對(duì)照組差異不顯著(P0.05),包囊減少可達(dá)到42.8%。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is an important opportunistic protozoa, widely parasitic in human and animal tissue cells, causing serious zoonosis.It is estimated that a third of the world's population is infected with the disease.At present, no ideal drug has been found to prevent and cure the disease. Due to the high cost of new drugs, the existence of drug residues and the strong virulence of attenuated vaccine, the research of Toxoplasma gondii genetic engineering vaccine has become a new trend.Through the study of Toxoplasma gondii antigen and its induced immune response, some scholars think that the effective vaccine has certain effect on the prevention and treatment of the disease.In order to investigate the immune protection of ADF gene against Toxoplasma gondii infection, primer was designed based on known ADF gene sequence, ADF open reading frame was amplified, cloned into prokaryotic expression vector and expressed recombinant ADF protein.The distribution of ADF gene in Toxoplasma gondii tachyzoites was located, and the ADF gene of Toxoplasma gondii was linked to the eukaryotic expression vector pVAX1 by DNA recombination technique, and the eukaryotic expression vector was constructed, and the recombinant eukaryotic expression plasmid was transfected into Hela cells.The recombinant ADF protein and the eukaryotic expression plasmid were immunized to test the immune protection of mice.The results showed that the ADF open reading frame of 365bp was amplified and the homology was 98 by Blast comparison, and the recombinant fusion protein of 17KDa was expressed. The ADF gene was located in the cytoplasm and plasma membrane of tachyzoites by immunofluorescence antibody technique.The specific protein was detected by indirect immunofluorescence assay in Hela cells transfected with recombinant plasmid, and the reactivity of the protein was confirmed by Western blot assay.With the increase of immunization times, the titer of antibody increased gradually, the difference between the test group and the control group was extremely significant (P 0.01). The number of CD8 T lymphocytes was significantly different from that of the control group. There was no significant difference in the number of CD 4 / CD 8 T lymphocytes between the test group and the control group (P 0.05).The experimental mice could not resist the infection of Toxoplasma gondii virulent strain, but the survival time of the test group was prolonged, the difference was not significant compared with the control group (P0.05), the number of the cyst in the brain tissue was decreased, and the worm reduction rate was 30%.Compared with the control group, the specific antibody titer (CD _ 4) and the number of CD8 T lymphocytes were significantly different between the test group and the control group. There was no significant difference in the number of CD _ 4 / CD _ 8 T lymphocytes between the test group and the control group (P 0.05).The survival time of the mice after attack was prolonged, which was significantly different from that of PBS control group (P 0.05) and pVAX1 control group (P 0.05), and the cysts decreased to 42.8%.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

【引證文獻(xiàn)】

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本文編號(hào)：1731331