Abstract:

The research was conducted to prepare the polyclonal antibody against goat estrogen receptor β (ERβ) and build the specific immunohistochemitry assay to detect ERβ in the goat’s ovary and uterus. The ERβ gene was amplified from Jining grey goat ovary by RT-PCR. The cloning sequence was identified by enzyme digestion and sequencing, then the gene was cloned into a prokaryotic expression vector pET32a(+) to construct recombinant plasmid named as pET32a(+)-ERβ. The expression of fusion protein was induced by IPTG in E. coli BL21( DE3) system and then identified by SDS-PAGE and Western blotting. After purification with Ni-NTA under denaturing condition, the fusion protein was applied as antigen to immunize New Zealand White rabbits for preparing specific polyclonal antibody. The titer of the antibody was detected by ELISA, and the specificity of the antibody was detected by Western blotting; the tissue sections of goat’s ovary and uterus were assayed with immunohistochemistry method mediated by the prepared antibody. The results showed the protein of ERβ was expressed as fusion protein, 53 kDa. Western blotting result indicated that ERβ fusion protein could specially reacted with polyclonal antibody against human ERβ. The titer of the antibody detected with ELISA was 1:2¹³, and the prepared antibody combined to ERβ was specific.the results of immunohistochemistry assay indicate ERβ mainly exists in the granule cells of ovary follicles and the epithelial cells and smooth muscle cells of the uterus endometrial layer. In conclusion, the polyclonal antibody against goat ERβ are successfully obtained in the present study and the ERβ distribution in the ovary and uterus of Jining Grey goat was firstly reported with immunohistochemistry assay mediated by the antibody against goat estrogen receptor β.