小瓜虫重组抑动抗原的亲和纯化及免疫学特性分析

发布时间：2018-04-23 07:20

本文选题：抑动抗原 + 小瓜虫　；参考：《福建农林大学》2008年硕士论文

【摘要】： 抑动抗原是小瓜虫(Ichthyophthirius multifiliis)的主要保护性抗原之一,在诱导宿主产生保护性免疫方面具有重要的作用,是研制小瓜虫疫苗的主要侯选抗原。本实验在构建G1株和G5株两种不同血清型小瓜虫抑动抗原基因重组四膜虫的基础上,通过优化培养重组四膜虫(Tetrahymena thermophila,共三株分别简称G1株,52A株和52B株),诱导重组四膜虫表达不同血清型的小瓜虫抑动抗原,采用非离子型去污剂Triton X-114提取虫体膜蛋白,通过亲和层析法纯化重组抑动抗原,采用ELISA、SDS-PAGE和Western-blotting分析重组抑动抗原的纯度及其免疫学特性。结果表明:①以小瓜虫特异性单克隆抗体为配体的亲和层析柱能够特异性地结合重组四膜虫所表达的抑动抗原蛋白,纯化的G1株和52B株表达产物与G1株和G5株小瓜虫抑动抗原的分子量和免疫原性相近,其分子量分别为44 kDa、45kDa(还原条件)或36 kDa、38kDa(非还原条件)。②重组抑动抗原与天然抑动抗原之间既有共同点,也存在部分差异。具体表现为:兔抗G1株重组抑动抗原(Tet-G1-iAg)血清能识别G1株和52B株重组抑动抗原,同时与非重组四膜虫(Neo)虫体抗原存在交叉反应;兔抗52B株重组抑动抗原(Tet-52B-iAg)血清不仅能识别同源的52A株、52B株重组抑动抗原,也能识别异源的G1株重组抑动抗原;而兔抗G1株小瓜虫抑动抗原(Ich-G1-iAg)血清能够识别所有供试的天然或重组表达抑动抗原,不与四膜虫虫体抗原产生任何交叉反应。③亲和纯化的重组抑动抗原(52B株)能诱导鳗鲡产生特异性的免疫应答,抗体水平峰值期为免疫后的20-30d。研究结果证明:以小瓜虫特异性单克隆抗体为配体的亲和层析柱可以用于小瓜虫重组抑动抗原的提取和纯化,且获得高度纯化的表达产物;血清学分析结果提示重组抑动抗原和天然抑动抗原免疫学特性基本一致;免疫试验证实重组抑动抗原能诱导鳗鲡产生特异性的免疫应答;上述研究结果为进一步开展小瓜虫疫苗学研究提供了有价值的实验依据和重要的技术基础。
[Abstract]:Inhibitory antigen is one of the main protective antigens of Ichthyophthirius multifiliis. It plays an important role in inducing host to produce protective immunity. In this study, we constructed two different serotypes, G1 and G5, and constructed the recombinant Tetrahymena quadrangea with different antigenic genes. By optimizing the culture of Tetrahymena thermophila, three strains of Tetrahymena thermophila were used to induce the recombinant Tetrahymena thermophila to express different antigens of different serotypes. The membrane protein was extracted by non-ionic decontamination agent Triton X-114. The recombinant inhibitory antigen was purified by affinity chromatography. The purity and immunological properties of the recombinant inhibitory antigen were analyzed by ELISA-SDS-PAGE and Western-blotting. The results showed that the affinity chromatography column with the specific monoclonal antibody of small melon worm as the ligand could specifically bind the inhibitory antigen protein expressed by the recombinant Tetrahymena. The molecular weight and immunogenicity of the purified G 1 and 52B strains were similar to those of G 1 and G 5 strains. Their molecular weights were 44 kDa 45kDa (reduction condition) or 36kDa (38kDa) respectively. There were some similarities and differences between the recombinant inhibitory antigen and the natural inhibitory antigen. The specific manifestations were as follows: the rabbit anti-G1 strain recombinant inhibitory antigen (Tet-G1-iAg) serum could recognize G1 strain and 52B strain recombinant inhibitory antigen, and there was cross-reaction with non-recombinant Tetrahymena tetrahymeni Neo) body antigen at the same time. Rabbit anti-52B strain recombinant inhibitory antigen (Tet-52B-iAg) serum could recognize not only the homologous 52A strain 52B strain recombinant inhibitory antigen, but also the heterologous G1 strain recombinant inhibitory antigen. However, the rabbit anti-G 1 strain Ich-G1-iAgA serum could recognize all natural or recombinant expressed inhibitory antigens. The recombinant inhibitory antigen 52B strain, which did not produce any cross reaction with Tetrahymena antigens, could induce specific immune response of Anguilla Anguilla japonica. The peak period of antibody level was 20-30 days after immunization. The results showed that the affinity chromatography column with specific monoclonal antibody could be used to extract and purify the recombinant inhibitory antigen, and the highly purified expression product could be obtained. The results of serological analysis showed that the immunological characteristics of recombinant inhibitory antigen and natural inhibitory antigen were basically the same, and the immunoassay showed that recombinant inhibitory antigen could induce specific immune response of Anguilla Anguilla japonica. These results provide valuable experimental basis and important technical basis for further research on small melon worm vaccine.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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