本文選題：多房棘球蚴病 + Em20.9�。� 參考：《甘肅農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：多房棘球蚴病又叫泡球蚴病,是一種重要的人獸共患寄生蟲病。在我國主要流行于西北牧區(qū),通常寄生于中間宿主肝、肺等臟器,嚴重危害動物和人體健康。目前主要采用化學(xué)藥物如阿苯達唑?qū)θM行定期驅(qū)蟲加以預(yù)防。該病潛伏期長,患者常常因此錯過最佳的治療時期,所以研制特異的早期診斷技術(shù)對該病的防治非常關(guān)鍵。多房棘球蚴20.9kD囊壁蛋白(Echinococcus multilocularis 20.9kD tegumental protein,Em20.9)是多房棘球絳蟲的一種表皮蛋白,其在蟲體與宿主的相互作用中發(fā)揮著重要作用,因此它有作為多房棘球蚴病的診斷和疫苗候選分子的潛力。本研究對多房棘球蚴的囊壁蛋白基因進行了克隆、表達、組織定位及ELISA檢測效果評價,主要研究結(jié)果如下:1.以多房棘球絳蟲cDNA為模板,擴增獲得多房棘球絳蟲Em20.9的CDS序列。生物信息學(xué)分析表明,Em20.9基因的ORF大小為543 bp,可編碼180個氨基酸,且具有多個抗原表位。2.構(gòu)建了原核表達重組質(zhì)粒pET28a-Em20.9,獲得了包涵體形式存在的重組蛋白rEm20.9,分子大小約26 ku。Western blotting分析表明,rEm20.9能被多房棘球蚴感染血清發(fā)生特異性識別。用純化后的rEm20.9免疫家兔,獲得了特異性的高滴度Ig G抗體。3.通過HE染色對多房棘球蚴進行形態(tài)學(xué)觀察,結(jié)果表明,一個多房棘球蚴囊泡中有許多處于不同發(fā)育階段的原頭蚴。免疫組化結(jié)果表明,Em20.9主要表達于多房棘球蚴的角皮層,說明多房棘球絳蟲的Em20.9可能參與到了與宿主的相互作用中。4.初步建立了以純化的Em20.9重組蛋白作為抗原的鼠多房棘球蚴病的間接ELISA檢測方法。通過方陣試驗確定了其最佳的操作條件為:最佳包被液為pH 9.6的碳酸鹽緩沖液(CBS),抗原的包被量為1μg/孔,一抗稀釋倍數(shù)為1:100,酶標(biāo)二抗的稀釋倍數(shù)為1:10 000,最佳封閉液為2.5%的BSA。確定臨界值為0.407,判定標(biāo)準(zhǔn)為OD450值≥0.437(cut-off+SD)為陽性,OD450值≤0.377(cut-off-SD)為陰性。將鼠多房棘球蚴病陽性和陰性血清倍比稀釋,當(dāng)稀釋倍數(shù)達到1:51 200時陽性血清OD450值仍大于判定標(biāo)準(zhǔn),說明具有良好的敏感性。同時對30份多房棘球蚴病陽性血清,50份陰性血清進行檢測,結(jié)果表明所建ELISA檢測方法的特異性為96.1%,敏感性為90.9%。
[Abstract]:Multilocular echinococcosis, also called alveolar echinococcosis, is an important zoonotic parasitic disease. It is mainly prevalent in northwest pastoral areas in China. It is usually parasitic on middle host liver, lung and other organs, which seriously endangers animal and human health. At present, chemical drugs such as albendazole are mainly used to prevent periodic insect repellent in dogs. Because of the long incubation period, patients often miss the best treatment period, so the development of specific early diagnosis technology is crucial to the prevention and treatment of the disease. Echinococcus multilocularis 20.9kD tegumental protein (Em20.9) is an epidermal protein of Echinococcus multilocularis 20.9kD tegumental, which plays an important role in the interaction between parasite and host, so it has the potential as a candidate molecule for the diagnosis and vaccine of Echinococcus multilocularis. In this study, the cyst wall protein gene of Echinococcus multilocularis was cloned, expressed, localized and evaluated by ELISA. The main results were as follows: 1. The CDS sequence of Em20.9 of Echinococcus multilocularis was amplified by using Echinococcus multilocularis cDNA as template. Bioinformatics analysis showed that the ORF size of Em20.9 gene was 543 BP, encoding 180 amino acids and having multiple epitopes. 2. A prokaryotic expression plasmid pET28a-Em20.9 was constructed, and the recombinant protein rEm20.9in the form of inclusion body was obtained. The molecular size of rEm20.9 was about 26 ku.Western blotting. The results showed that rEm20.9 could be specifically recognized by the sera of Echinococcus multilocularis infection. After immunizing rabbits with purified rEm20.9, a specific high titer IgG antibody. 3. Morphological observation of hydatid multilocularis was carried out by HE staining. The results showed that there were many protocariae in one alveolar of echinococcus multilocularis at different stages of development. The immunohistochemical results showed that Em20.9 was mainly expressed in the keratoderma of Echinococcus multilocularis, suggesting that the Em20.9 of Echinococcus multilocularis might be involved in the interaction with host. An indirect ELISA method for the detection of murine echinococcosis using purified Em20.9 recombinant protein as antigen was established. The optimum operating conditions were determined by square array test: the best coating solution was carbonate buffer solution with pH 9.6, the encapsulation amount of antigen was 1 渭 g / pore, the dilution multiple of the first antibody was 1: 100, the dilution multiple of the enzyme labeled second antibody was 1:10, and the best sealing solution was BSA with 2.5%. The critical value was determined to be 0.407, and the positive OD450 value 鈮，

本文編號：1958070