贾第虫贾第素蛋白多克隆抗体的制备及免疫荧光定位研究

发布时间：2018-06-03 12:14

本文选题：十二指肠贾第鞭毛虫 + 原核表达　；参考：《东北农业大学》2017年硕士论文

【摘要】：贾第虫病(giardiasis)自上世纪70年代末已被世界卫生组织列为人兽共患寄生虫病。十二指肠贾第虫(Giardia duodenalis)是通过粪-口途径传播并在脊椎动物中引起腹泻的重要寄生虫,能够广泛感染人、家畜及野生动物。此外,贾第虫还可感染鱼、两栖动物、爬行动物、鸟类和啮齿动物。贾第虫感染人常引起腹泻,同时伴有胃肠胀气、腹痛、肠病变、体重减轻和吸收不良综合征等症状,儿童、免疫缺陷或受损的人最易感。对于家畜,贾第虫可导致其发病从而造成相应的经济损失。贾第虫也是旅行者腹泻常见的病原。因此,贾第虫病被视为重要的人兽共患寄生虫病。预防贾第虫病的关键是建立灵敏特异的诊断方法,对病原快速有效的鉴定对于研究和了解贾第虫的流行病学至关重要。经研究发现,贾第虫细胞骨架蛋白和毒力之间存在较强的生物学联系。因为贾第虫必须借助其细胞骨架在肠内移行并附着于肠黏膜上以避免被清除。贾第虫是高度分化的原生动物,细胞骨架主要包括四对鞭毛、腹吸盘和中体等结构。贾第素是贾第虫特有的、含量丰富的细胞骨架蛋白,其已被划分为α-贾第素(α-giardin),β-贾第素(β-giardin),δ-贾第素(δ-giardin)和γ-贾第素(γ-giardin),大小在29-38 k Da。α-giardins是一类钙依赖性膜结合蛋白,包含21个成员的多基因家族。α-giardins的多样性揭示了贾第虫生物学功能的重要性。目前,国内外对α-giardins的研究较多,但对δ-giardin和γ-giardin的研究相对较少。因此,本研究根据Gen Bank中公布的δ-giardin和γ-giardin序列设计引物,从贾第虫集聚体A中提取了总RNA,反转录得到c DNA。以c DNA为模板扩增了δ-giardin和γ-giardin目的基因,成功构建了重组表达质粒p ET-28a-δ-giardin和p ET-28a-γ-giardin,将其转化到感受态细胞Rosseta(DE3)。经IPTG诱导进行重组蛋白的原核表达。再经过KCl染色切胶纯化法获得较纯的融合蛋白,以不同浓度梯度的尿素作为变性剂,溶解包涵体进行透析复性。随后,针对重组蛋白制备了多克隆抗体,并且利用免疫荧光定位技术分析贾第素在虫体中的分布情况。结果表明,δ-giardin基因全长882 bp,编码293个氨基酸;γ-giardin基因全长936bp,编码311个氨基酸,重组蛋白相对分子量分别约为36 k Da和38 k Da,重组蛋白均以包涵体的形式存在。然而,试验过程中,δ-giardin表达量较低,纯化后的浓度不适于免疫动物。ELISA和Western blot实验鉴定γ-giardin抗体具有良好的抗原特异性与优异的抗原结合活性。免疫荧光定位技术表明其主要位于贾第虫滋养体的腹吸盘。本试验旨在为建立ELISA检测方法提供理论依据,为今后探讨贾第虫致病机制、临床诊断和治疗方面提供理论基础。
[Abstract]:Giardiasis has been listed as zoonotic parasitic disease by the World Health Organization (WHO) since the late 1970s. Giardia duodenalis (Giardia duodenalis) is an important parasite that is transmitted by fecal-oral route and causes diarrhea in vertebrates. It can infect people, livestock and wild animals widely. In addition, Giardia can infect fish, amphibians, reptiles, birds and rodents. Giardia infection is often associated with diarrhea accompanied by gastrointestinal flatulence, abdominal pain, intestinal lesions, weight loss and poor absorption syndrome. Children, immunodeficient or impaired people are most susceptible. For domestic animals, Giardia can cause disease, resulting in corresponding economic losses. Giardia is also a common cause of traveler's diarrhea. Therefore, giardiosis is regarded as an important zoonotic parasitic disease. The key to prevent giardiasis is to establish a sensitive and specific diagnostic method. Rapid and effective identification of the pathogen is very important for the study and understanding of the epidemiology of giardia. It was found that there was a strong biological relationship between the cytoskeleton protein and virulence of Giardia lamblia. Because Giardia must use its cytoskeleton to migrate in the intestine and adhere to the intestinal mucosa to avoid clearance. Giardia giardia is a highly differentiated protozoan. The cytoskeleton consists of four pairs of flagellum, abdominal sucker and midbody. It has been classified as 伪 -giardinine, 尾 -giardinine, 未 -giardinin and 纬 -giardinin, which is a calcium dependent membrane binding protein at 29-38 k Da. 伪 -giardins. The diversity of 伪 -giardins revealed the importance of biological function of Giardia lamblia. At present, there are many studies on 伪 -giardins at home and abroad, but relatively few on 未 -giardin and 纬 -giardin. Therefore, based on the 未 -giardin and 纬 -giardin sequences published in Gen Bank, the total RNAs were extracted from Giardia lamblia agglomeration A, and c DNAs were obtained by reverse transcription. The target genes of 未 -giardin and 纬 -giardin were amplified by using c DNA as template. The recombinant expression plasmids pET-28a- 未 -giardin and pET-28a- 纬 -giardinwere successfully constructed and transformed into Rossetade3. The prokaryotic expression of recombinant protein was induced by IPTG. The fusion protein was purified by KCl staining, and the inclusion body was dissolved and renatured by dialysis with urea with different concentration gradient as denaturant. Subsequently, polyclonal antibodies were prepared against the recombinant protein, and the distribution of Jardine in the parasites was analyzed by immunofluorescence localization technique. The results showed that the length of 未 -giardin gene was 882 BP, encoding 293 amino acids, and 纬 -giardin gene was 936 BP, encoding 311 amino acids. The relative molecular weight of the recombinant protein was about 36 kDa and 38 k, respectively. However, the expression of 未 -giardin was low, and the purified concentration was not suitable for immunizing animals. Elisa and Western blot assay showed that 纬 -giardin antibody had good antigen-specificity and excellent antigen-binding activity. Immunofluorescence localization technique showed that it was mainly located in the abdominal sucker of Giardia japonica trophozoites. The purpose of this study was to provide a theoretical basis for the establishment of ELISA detection methods, and to provide theoretical basis for the further study of the pathogenesis, clinical diagnosis and treatment of Giardia lamblia.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4

【参考文献】