【摘要】：豬囊尾蚴病(以下簡稱囊蟲病)是人獸共患寄生蟲病,病原體為豬帶絳蟲(Taenia solium)的幼蟲豬囊尾蚴。囊蟲病的危害程度,因寄生部位和數(shù)量而有所不同,其中以腦囊蟲病危害最大。囊尾蚴病的有效診斷是控制該病的關(guān)鍵。然而,作為一種寄生于人、豬等脊椎動物體內(nèi)的扁形動物門寄生蟲,其抗原成分復雜,且與其他帶科寄生蟲抗原間存在廣泛交叉性,因此,囊尾蚴有效診斷抗原的篩選工作難度大、任務(wù)繁重。從組織結(jié)構(gòu)上來說,囊尾蚴抗原包括囊液抗原、囊壁抗原和頭節(jié)抗原,且這些抗原隨囊尾蚴的發(fā)育階段不同而呈現(xiàn)出變化。另外,囊尾蚴在宿主體內(nèi)生長發(fā)育過程中還會排泄分泌到其體外一些抗原成分,即排泄分泌抗原(excretary-secretary antigen,ES-Ag)。盡管如此,隨著分子生物學的發(fā)展,囊尾蚴重組診斷抗原的研究仍是免疫診斷的熱點。與囊尾蚴囊壁、頭節(jié)相比,囊尾蚴囊液的糖蛋白和脂蛋白含量豐富,且其抗原特異性較高,抗原成分的pH值主要位于堿性區(qū)。另外,研究顯示當人體感染豬囊尾蚴后,幾乎所有患者血清中特異IgG4顯著性升高,其含量約占總反應(yīng)IgG抗體50%以上,因此檢測囊蟲病患者血清特異IgG4具有較好的敏感性,特別是IgG4為短程抗體,當患者體內(nèi)的囊尾蚴被殺滅后,特異IgG4會較快的降低或消失,客服了檢測總IgG不能進行療效考核的不足。本實驗利用收集到的囊尾蚴囊液抗原進行雙向電泳和免疫印記,發(fā)現(xiàn)與IgG4抗體結(jié)合力較強的蛋白以堿性居多,以小分子量蛋白為主。經(jīng)抗原篩選出51個抗原點,對抗原點進行質(zhì)譜分析,找出24個陽性點。24個陽性點與12種囊蟲蛋白密切相關(guān),每個陽性點都有4到5個相關(guān)蛋白(分值大于等于39),其中最為密切相關(guān)的蛋白有4種。分別為Secreted antigen Ts8B3(16/24),Secreted antigen Ts8B2(18/24),Sequence 3 from patent US 6589752(15/24)和Cysticercosis 10kDa antigen(16/24)。選取其中排泄分泌抗原Ts8B3基因,并將成熟肽序列亞克隆到載體中,在大腸桿菌細胞中成功表達了重組融合蛋白。通過ELISA鑒定,重組Ts8B3抗原能很好的與IgG4結(jié)合。
[Abstract]:Cysticercosis (hereinafter referred to as cysticercosis) is a zoonotic parasitic disease, and the pathogen is cysticercaria cellulosae of (Taenia solium). The degree of damage of cysticercosis varies according to the parasitic site and quantity, among which cerebral cysticercosis is the most harmful. The effective diagnosis of cysticercosis is the key to control the disease. However, as a parasite of phylum oblate in vertebrates such as human beings, pigs and other vertebrates, its antigenic components are complex and widely intersected with other parasitic antigens of the family. Screening for effective antigen diagnosis of cysticercaria is difficult and arduous. In terms of tissue structure, cysticercus antigen includes cystic fluid antigen, cyst wall antigen and head ganglion antigen, and these antigens vary with the development stage of cysticercus. In addition, Cysticercus cysticercus excreted and secreted some antigens in vitro, I. e., excretory secretory antigen (excretary-secretary antigen,ES-Ag) during the growth and development of cysticercus in vivo. However, with the development of molecular biology, the study of recombinant diagnostic antigen of cysticercus is still a hot topic in immunological diagnosis. Compared with the cyst wall and head ganglion of cysticercus, the contents of glycoprotein and lipoprotein in cyst fluid of cysticercus were rich, and its antigen specificity was higher. The pH value of antigen component was mainly located in alkaline region. In addition, the study showed that the specific IgG4 content in the sera of almost all patients increased significantly after infection with cysticercus cellulosae, which accounted for more than 50% of the total reactive IgG antibody. Therefore, the detection of serum specific IgG4 in patients with cysticercosis had a good sensitivity. Especially, IgG4 is a short course antibody. When the cysticercus is killed, the specific IgG4 will decrease or disappear quickly. In this experiment, the collected cyst fluid antigen of cysticercus cellulosae was detected by two dimensional electrophoresis and imprinting. It was found that most of the proteins with strong binding ability to IgG4 antibody were alkaline, and most of the proteins were small molecular weight proteins. 51 antigenic sites were screened by antigen, and 24 positive spots were found by mass spectrometry analysis. 24 positive spots were closely related to 12 cysticercosis proteins, and each positive spot had 4 to 5 related proteins (the score was greater than 39). Among them, there are 4 kinds of proteins most closely related. Secreted antigen Ts8B3 (16 / 24), Secreted antigen Ts8B2) and Cysticercosis 10kDa antigen (18 / 24), Sequence 3 from patent US 6589752) and Cysticercosis 10kDa antigen (16 / 24), respectively. The excretory secretory antigen Ts8B3 gene was selected, and the mature peptide sequence was subcloned into the vector. The recombinant fusion protein was successfully expressed in Escherichia coli cells. By ELISA identification, the recombinant Ts8B3 antigen can bind well with IgG4.
【學位授予單位】：濟南大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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