多房棘球蚴20.9kD囊壁蛋白基因的原核表达、组织定位及ELISA检测方法的建立

发布时间：2018-05-31 03:16

  本文选题：多房棘球蚴病 + Em20.9　； 参考：《甘肃农业大学》2017年硕士论文

【摘要】：多房棘球蚴病又叫泡球蚴病,是一种重要的人兽共患寄生虫病。在我国主要流行于西北牧区,通常寄生于中间宿主肝、肺等脏器,严重危害动物和人体健康。目前主要采用化学药物如阿苯达唑对犬进行定期驱虫加以预防。该病潜伏期长,患者常常因此错过最佳的治疗时期,所以研制特异的早期诊断技术对该病的防治非常关键。多房棘球蚴20.9kD囊壁蛋白(Echinococcus multilocularis 20.9kD tegumental protein,Em20.9)是多房棘球绦虫的一种表皮蛋白,其在虫体与宿主的相互作用中发挥着重要作用,因此它有作为多房棘球蚴病的诊断和疫苗候选分子的潜力。本研究对多房棘球蚴的囊壁蛋白基因进行了克隆、表达、组织定位及ELISA检测效果评价,主要研究结果如下:1.以多房棘球绦虫cDNA为模板,扩增获得多房棘球绦虫Em20.9的CDS序列。生物信息学分析表明,Em20.9基因的ORF大小为543 bp,可编码180个氨基酸,且具有多个抗原表位。2.构建了原核表达重组质粒pET28a-Em20.9,获得了包涵体形式存在的重组蛋白rEm20.9,分子大小约26 ku。Western blotting分析表明,rEm20.9能被多房棘球蚴感染血清发生特异性识别。用纯化后的rEm20.9免疫家兔,获得了特异性的高滴度Ig G抗体。3.通过HE染色对多房棘球蚴进行形态学观察,结果表明,一个多房棘球蚴囊泡中有许多处于不同发育阶段的原头蚴。免疫组化结果表明,Em20.9主要表达于多房棘球蚴的角皮层,说明多房棘球绦虫的Em20.9可能参与到了与宿主的相互作用中。4.初步建立了以纯化的Em20.9重组蛋白作为抗原的鼠多房棘球蚴病的间接ELISA检测方法。通过方阵试验确定了其最佳的操作条件为:最佳包被液为pH 9.6的碳酸盐缓冲液(CBS),抗原的包被量为1μg/孔,一抗稀释倍数为1:100,酶标二抗的稀释倍数为1:10 000,最佳封闭液为2.5%的BSA。确定临界值为0.407,判定标准为OD450值≥0.437(cut-off+SD)为阳性,OD450值≤0.377(cut-off-SD)为阴性。将鼠多房棘球蚴病阳性和阴性血清倍比稀释,当稀释倍数达到1:51 200时阳性血清OD450值仍大于判定标准,说明具有良好的敏感性。同时对30份多房棘球蚴病阳性血清,50份阴性血清进行检测,结果表明所建ELISA检测方法的特异性为96.1%,敏感性为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，

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