本文關(guān)鍵詞：小鼠SMCY重組蛋白及抗H-Y抗原抗體IgY的純化　出處：《上海交通大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： H-Y SMCY重組蛋白 Ni-NTA 親和層析 IgY

【摘要】： 提取經(jīng)雄性動(dòng)物細(xì)胞免疫后的雌性動(dòng)物抗血清,得到的抗體純度不高、效價(jià)太低,無法用于H-Y抗原的進(jìn)一步研究。本研究以SMCY重組蛋白代替雄性動(dòng)物細(xì)胞作為免疫材料,免疫隱性白母雞,收集雞蛋,PEG 6000方法提取蛋黃中的IgY抗體。為了進(jìn)一步得到高純度的特異性抗H-Y抗原抗體,我們將Ni-NTA柱純化N端帶有6his-tag標(biāo)簽的SMCY重組蛋白作為配基,偶聯(lián)至CNBr活化的Sepharose 4B制成親和層析柱,分離純化抗H-Y抗原的特異性抗體IgY。結(jié)果表明,經(jīng)Ni-NTA柱純化后,SMCY重組蛋白純度從28%達(dá)到80%以上,制作成的親和層析柱純化可以得到純度高達(dá)97%的抗H-Y抗原的特異性抗體IgY。經(jīng)雌、雄小鼠肝臟細(xì)胞的免疫組化檢測(cè),熒光顯微鏡下能夠觀察到雄性小鼠肝臟細(xì)胞有強(qiáng)烈熒光,而在雌小鼠肝臟細(xì)胞表面觀察不到特異熒光。研究結(jié)果表明,SMCY重組蛋白具有H-Y抗原的免疫原性,能夠代替雄性動(dòng)物細(xì)胞作為免疫材料;Ni-NTA柱純化后的SMCY重組蛋白,可作為親和配基制作親和層析柱,用于純化分離抗H-Y抗原的特異性抗體;IgY技術(shù)生產(chǎn)的卵黃抗體中有較高含量的抗H-Y抗原的特異性抗體;親和層析純化以后,得到的IgY抗體能夠特異識(shí)別結(jié)合與雄性小鼠肝臟細(xì)胞表面,對(duì)H-Y抗原具有很好的特異性。
[Abstract]:The purity and titer of the antiserum extracted from female animals immunized by male animal cells were not high and the titer was too low. This study used SMCY recombinant protein instead of male animal cells as immune material to immunize recessive white hens and collect eggs. PEG 6000 method was used to extract IgY antibody from egg yolk. In order to obtain high purity anti-H-Y antigen antibody. Ni-NTA column was used to purify N-terminal SMCY recombinant protein with 6his-tag tag as ligand and then coupled to CNBr activated Sepharose 4B to prepare affinity chromatography column. The specific antibody against H-Y antigen was isolated and purified. The results showed that the purity of SMCY recombinant protein was above 80% from 28% after purification by Ni-NTA column. Purified by affinity chromatography column, the specific antibody of anti-H-Y antigen with purity up to 97% was obtained. The immunocytochemistry of liver cells of female and male mice was carried out. Strong fluorescence was observed in the liver cells of male mice under fluorescence microscope, but no specific fluorescence was observed on the surface of liver cells in female mice. SMCY recombinant protein has the immunogenicity of H-Y antigen and can replace male animal cells as immune material. The recombinant SMCY protein purified by Ni-NTA column can be used as affinity ligand to prepare affinity chromatography column for purification of specific antibodies against H-Y antigen. The yolk antibody produced by IgY had a high content of specific antibody against H-Y antigen. After purification by affinity chromatography, the obtained IgY antibody could specifically recognize and bind to the surface of the liver cells of male mice, and had a good specificity for H-Y antigen.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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