抗猪囊尾蚴特异IgG4重组抗原的制备与鉴定

发布时间：2019-01-07 13:47

【摘要】：猪囊尾蚴病(以下简称囊虫病)是人兽共患寄生虫病,病原体为猪带绦虫(Taenia solium)的幼虫猪囊尾蚴。囊虫病的危害程度,因寄生部位和数量而有所不同,其中以脑囊虫病危害最大。囊尾蚴病的有效诊断是控制该病的关键。然而,作为一种寄生于人、猪等脊椎动物体内的扁形动物门寄生虫,其抗原成分复杂,且与其他带科寄生虫抗原间存在广泛交叉性,因此,囊尾蚴有效诊断抗原的筛选工作难度大、任务繁重。从组织结构上来说,囊尾蚴抗原包括囊液抗原、囊壁抗原和头节抗原,且这些抗原随囊尾蚴的发育阶段不同而呈现出变化。另外,囊尾蚴在宿主体内生长发育过程中还会排泄分泌到其体外一些抗原成分,即排泄分泌抗原(excretary-secretary antigen,ES-Ag)。尽管如此,随着分子生物学的发展,囊尾蚴重组诊断抗原的研究仍是免疫诊断的热点。与囊尾蚴囊壁、头节相比,囊尾蚴囊液的糖蛋白和脂蛋白含量丰富,且其抗原特异性较高,抗原成分的pH值主要位于碱性区。另外,研究显示当人体感染猪囊尾蚴后,几乎所有患者血清中特异IgG4显著性升高,其含量约占总反应IgG抗体50%以上,因此检测囊虫病患者血清特异IgG4具有较好的敏感性,特别是IgG4为短程抗体,当患者体内的囊尾蚴被杀灭后,特异IgG4会较快的降低或消失,客服了检测总IgG不能进行疗效考核的不足。本实验利用收集到的囊尾蚴囊液抗原进行双向电泳和免疫印记,发现与IgG4抗体结合力较强的蛋白以碱性居多,以小分子量蛋白为主。经抗原筛选出51个抗原点,对抗原点进行质谱分析,找出24个阳性点。24个阳性点与12种囊虫蛋白密切相关,每个阳性点都有4到5个相关蛋白(分值大于等于39),其中最为密切相关的蛋白有4种。分别为Secreted antigen Ts8B3(16/24),Secreted antigen Ts8B2(18/24),Sequence 3 from patent US 6589752(15/24)和Cysticercosis 10kDa antigen(16/24)。选取其中排泄分泌抗原Ts8B3基因,并将成熟肽序列亚克隆到载体中,在大肠杆菌细胞中成功表达了重组融合蛋白。通过ELISA鉴定,重组Ts8B3抗原能很好的与IgG4结合。
[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.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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