小鼠smcy重组蛋白及抗h-y抗原抗体igy的纯化-purification of mouse smcy recombinant protein and anti - h - y antigen antibody igy.docxVIP

THE PURIFICATION of MOUSE SMCY RECOMBINED PROTEIN AND IgY Abstract In the previous study, anti-H-Y antibody was extracted from the serum of female animals which were immunized with male cells. But it can’t be direct used for the further study because of its low purity and titer. In the present study, a new purification method was applied. We immunized Recessive White hens with SMCY recombined protein, and then extracted antibody IgY from the egg yolk with PEG 6000. Then the SMCY recombined protein was purified with Ni-NTA chromatography with its 6 his-tag at the N-terminal, and then it was combined to Sepharose 4B activated by CNBr in order to get the anti-H-Y antibody. After purification, the purity of SMCY recombined protein achieved to high level more than 80%, and the specified IgY antibody achieved to 97%. By IgY antibody immunohistochemical assay, we could see bright fluorescence from the male mouse liver cells under the fluorescence microscope while almost no fluorescence on the surface of female cells. The study showed that SMCY recombined protein had the same immunogenicity as H-Y antigen, which made it a good substitute for male cells as immune materials. After purification, the SMCY recombined protein could be used as an immune ligand in the purification of anti-H-Y antibody IgY, which was contained with a high concentration in the antibody from egg yolk produced through IgY technology. And our purified anti-H-Y antibody showed a good specific character on recognizing the liver cells of male mouse. Keywords ： H-Y, SMCY recombined protein, Ni-NTA, affinity chromatography, IgY 目 录 第 1 章 绪 论 9 HYPERLINK \l _TOC_250007 1 H-Y 抗原的研究现状 9 HYPERLINK \l _TOC_250006 1.1 H-Y 抗原的血清学研究 9 HYPERLINK \l _TOC_250005 1.2 H-Y 单克隆抗体的研究 10 HYPERLINK \l _TOC_250004 1.3 H-Y 抗原相应基因的研究 10 HYPERLINK \l _TOC_250003 2 IGY 技术 11 HYPERLINK \l _TOC_250002 2.1 IgY 的分子结构及基本性质 11 HYPERLINK \l _TOC_250001 2.2 免疫鸡产生 IgY 的技术 11 HYPERLINK \l _TOC_250000 2.3 IgY 的提取和纯化技术 12 3