小鼠SMCY重组蛋白及抗H-Y抗原抗体IgY的纯化

发布时间：2018-01-04 09:44

本文关键词：小鼠SMCY重组蛋白及抗H-Y抗原抗体IgY的纯化　出处：《上海交通大学》2008年硕士论文　论文类型：学位论文

更多相关文章： H-Y SMCY重组蛋白 Ni-NTA 亲和层析 IgY

【摘要】： 提取经雄性动物细胞免疫后的雌性动物抗血清,得到的抗体纯度不高、效价太低,无法用于H-Y抗原的进一步研究。本研究以SMCY重组蛋白代替雄性动物细胞作为免疫材料,免疫隐性白母鸡,收集鸡蛋,PEG 6000方法提取蛋黄中的IgY抗体。为了进一步得到高纯度的特异性抗H-Y抗原抗体,我们将Ni-NTA柱纯化N端带有6his-tag标签的SMCY重组蛋白作为配基,偶联至CNBr活化的Sepharose 4B制成亲和层析柱,分离纯化抗H-Y抗原的特异性抗体IgY。结果表明,经Ni-NTA柱纯化后,SMCY重组蛋白纯度从28%达到80%以上,制作成的亲和层析柱纯化可以得到纯度高达97%的抗H-Y抗原的特异性抗体IgY。经雌、雄小鼠肝脏细胞的免疫组化检测,荧光显微镜下能够观察到雄性小鼠肝脏细胞有强烈荧光,而在雌小鼠肝脏细胞表面观察不到特异荧光。研究结果表明,SMCY重组蛋白具有H-Y抗原的免疫原性,能够代替雄性动物细胞作为免疫材料;Ni-NTA柱纯化后的SMCY重组蛋白,可作为亲和配基制作亲和层析柱,用于纯化分离抗H-Y抗原的特异性抗体;IgY技术生产的卵黄抗体中有较高含量的抗H-Y抗原的特异性抗体;亲和层析纯化以后,得到的IgY抗体能够特异识别结合与雄性小鼠肝脏细胞表面,对H-Y抗原具有很好的特异性。
[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.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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