Preparation and Application of Yak (Bos grunniens) HMGN2 Polyclonal Antibody
JIA Li-Yi1, PAN Yang-Yang1,2, WANG Meng1,2, ZHANG Hui1, LI Yu-Ping1, XU Zi-Jiang1, WANG Jun-Qian1, YU Si-Jiu1,2, WANG Li-Bin1,2,*
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 Gansu Province Livestock Embryo Engineering Research Center, Lanzhou 730070, China
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.
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