發(fā)布時間：2018-04-23 07:20

  本文選題：抑動抗原 + 小瓜蟲　； 參考：《福建農(nóng)林大學(xué)》2008年碩士論文

【摘要】： 抑動抗原是小瓜蟲(Ichthyophthirius multifiliis)的主要保護(hù)性抗原之一,在誘導(dǎo)宿主產(chǎn)生保護(hù)性免疫方面具有重要的作用,是研制小瓜蟲疫苗的主要侯選抗原。本實驗在構(gòu)建G1株和G5株兩種不同血清型小瓜蟲抑動抗原基因重組四膜蟲的基礎(chǔ)上,通過優(yōu)化培養(yǎng)重組四膜蟲(Tetrahymena thermophila,共三株分別簡稱G1株,52A株和52B株),誘導(dǎo)重組四膜蟲表達(dá)不同血清型的小瓜蟲抑動抗原,采用非離子型去污劑Triton X-114提取蟲體膜蛋白,通過親和層析法純化重組抑動抗原,采用ELISA、SDS-PAGE和Western-blotting分析重組抑動抗原的純度及其免疫學(xué)特性。結(jié)果表明:①以小瓜蟲特異性單克隆抗體為配體的親和層析柱能夠特異性地結(jié)合重組四膜蟲所表達(dá)的抑動抗原蛋白,純化的G1株和52B株表達(dá)產(chǎn)物與G1株和G5株小瓜蟲抑動抗原的分子量和免疫原性相近,其分子量分別為44 kDa、45kDa(還原條件)或36 kDa、38kDa(非還原條件)。②重組抑動抗原與天然抑動抗原之間既有共同點,也存在部分差異。具體表現(xiàn)為:兔抗G1株重組抑動抗原(Tet-G1-iAg)血清能識別G1株和52B株重組抑動抗原,同時與非重組四膜蟲(Neo)蟲體抗原存在交叉反應(yīng);兔抗52B株重組抑動抗原(Tet-52B-iAg)血清不僅能識別同源的52A株、52B株重組抑動抗原,也能識別異源的G1株重組抑動抗原;而兔抗G1株小瓜蟲抑動抗原(Ich-G1-iAg)血清能夠識別所有供試的天然或重組表達(dá)抑動抗原,不與四膜蟲蟲體抗原產(chǎn)生任何交叉反應(yīng)。③親和純化的重組抑動抗原(52B株)能誘導(dǎo)鰻鱺產(chǎn)生特異性的免疫應(yīng)答,抗體水平峰值期為免疫后的20-30d。 研究結(jié)果證明:以小瓜蟲特異性單克隆抗體為配體的親和層析柱可以用于小瓜蟲重組抑動抗原的提取和純化,且獲得高度純化的表達(dá)產(chǎn)物;血清學(xué)分析結(jié)果提示重組抑動抗原和天然抑動抗原免疫學(xué)特性基本一致;免疫試驗證實重組抑動抗原能誘導(dǎo)鰻鱺產(chǎn)生特異性的免疫應(yīng)答;上述研究結(jié)果為進(jìn)一步開展小瓜蟲疫苗學(xué)研究提供了有價值的實驗依據(jù)和重要的技術(shù)基礎(chǔ)。
[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.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

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本文編號：1790990