【摘要】 高迁移率族核小体结合域蛋白2 (high mobility group nucleosomal binding domain 2, HMGN2)在哺乳动物胚胎发育过程中高度表达，可与核小体结合，改变染色体结构，参与调节细胞分化、胚胎发育等过程，为探究HMGN2蛋白在牦牛(Bos grunniens)子宫中的作用机理，本研究以牦牛为研究对象，克隆牦牛HMGN2基因并制备牦牛HMGN2多克隆抗体，通过qPCR检测HMGN2基因在牦牛不同时期子宫中的相对表达量；利用纯化的牦牛HMGN2重组蛋白免疫新西兰大白兔(Oryctolagus cuniculus)，通过A/G-Sepharose柱层析法纯化制备牦牛HMGN2多克隆抗体；利用Western blot检测HMGN2蛋白的表达，并通过免疫组织化学法(immunohistochemistry, IHC)检测HMGN2蛋白的定位。结果表明，本研究成功克隆牦牛HMGN2基因(GenBank No. OR713742)并构建重组质粒pKMD-SUMO-HMGN2，诱导产生重组蛋白，符合预期结果。qPCR和Western blot的结果显示，HMGN2基因及蛋白在牦牛妊娠早期子宫中的表达量最高。IHC检测结果表明，HMGN2主要表达于子宫腺与子宫内膜。提示HMGN2可能在牦牛妊娠早期对胚胎的附植和胎儿的生长发育具有重要调节作用。以上结果表明，本研究所制备的牦牛HMGN2抗体可为进一步研究HMGN2蛋白的作用机理提供基础资料。更多还原

【Abstract】 High mobility group nucleosomal binding domain 2(HMGN2) is highly expressed in mammalian embryonic development, which can bind to nucleosomes, change chromosome structure, and participate in the regulation of cell differentiation and embryonic development. In order to explore the mechanism of HMGN2 protein in the uterus of yak(Bos grunniens), in this study, yak HMGN2 gene was cloned and HMGN2 polyclonal antibody was prepared, and the relative expression of HMGN2 gene in yak uterus at different stages was detected by qPCR. Oryctolagus cuniculus was immunized with purified yak HMGN2 recombinant protein,and yak HMGN2 polyclonal antibody was purified by A/G-Sepharose column chromatography. Western blot was used to detect the expression of HMGN2 protein, and immunohistochemistry(IHC) was used to detect the localization of HMGN2 protein. The results showed that the recombinant protein was induced by the successful cloning of yak HMGN2 gene(GenBank No. OR713742) and the construction of recombinant plasmid pKMD-SUMO-HMGN2 in this study were in line with the expected results. The results of qPCR and Western blot showed that HMGN2 gene and protein were the highest in the first trimester of yak uterus. The results of IHC showed that HMGN2 was mainly expressed in the uterine gland and endometrium suggesting that HMGN2 might play a regulatory role in embryo implantation and fetal growth and development in the early pregnancy of yak uterus. These results indicated that the yak HMGN2 antibody prepared in this study could be used for molecular experiments using yak as a model, which provides basic data for further study of the mechanism of action of HMGN2 protein.更多还原