Cloning Of ERα And ERβ Complete Genes In Jining Grey Goats And Yimeng Black Goats And Preparation Of Polyclonal Antibodies And Theirs Application

Jining Grey goats, widely distributing in Jining and Heze regions in Shandong Provinceof China, are noted for the wavy patterns of its kid-pelt which is the traditional commodity ininternational markets and display significant performances of sexual precocity. Yimeng Blackgoats which locate in the southwest of ShanDong are characteristic of common sexualmaturity, low fecund ability. Their reproductive traits are obvious different. The geneticmechanism of their different biological performances is interesting secientific problem. It isrelated with expression, distribution and function or not has not been reported. At present, twosubtypes of ERs gene in Jining Grey goats and Yimeng Black goats were not been published.In addition, due to the lack of specificity antibody, it is impossible to conduct farther study ontheir ERs distribution, expression and biological function.In this study, the ERα and ERβ genes were amplified from Jining grey goat ovary byRT-PCR and identified by enzyme digestion and sequencing, then the genes were cloned intoa prokaryotic expression vector pET32a(+) to construct recombinant plasmid named aspET32a(+)-ERα and pET32a(+)-ERβ. The expression of fusion proteins were induced byIPTG 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 proteins were appliedas antigen to immunize New Zealand White rabbits for preparing specific polyclonal antibody.The titer of the antibodies were detected by ELISA, and the specificity of the antibodies weredetected by Western blotting; the tissue sections of goat’s ovary and uterus were assayed withimmunohistochemistry method mediated by the prepared antibody. The results showed:1. The genes of ERα and ERβ in Jining Grey goats and Yimeng Black goats wasamplified with completed open-reading-frame,which includes1791bp and1584bp sequencesrespectively.Compared with that of other animals’ species and constructing phylogenetic trees,Estrogen receptor genes is highly conservative in species evolution process. The two goats estrogen receptor amino acid sequences and protein spatial structure was compared with otheranimals’, the variation of ERβ was more than ERα.2. The prokaryotic expression vector pET32a(+)-ERα and pET32a(+)-ERβ wereconstruct and the proteins of ERα and ERβ were expressed as fusion protein,45ku and53ku.Western blotting result indicated that the fusion proteins could specially reacted withpolyclonal antibody against human ERs. The titers of the antibodies detected with ELISAwere both1:214, and the prepared antibodies combined to ERs were specific.3. The immunohistochemical methods mediated with ERα and ERβ antibodies were setup and used to detect two subtypes of estrogen receptor distribution. ERα mainly exists in thegranule cells and theca cells of ovary follicles and the epithelial cells, glandular epitheliumand smooth muscle cells of the uterus endometrial layer and ERβ mainly exists in the granulecells of ovary follicles and the epithelial cells and smooth muscle cells of the uterusendometrial layer in Jining Grey goats and Yimeng Black goats.In conclusion, the completed genes with ORF of ERα and ERβ in Jining Grey goats andYimeng Black goats successfully were cloned and the polyclonal antibody against goat ERsare prepared in the present study. ERα and ERβ distribution in the ovary and uterus of JiningGrey goat were reported with immunohischemistry assay mediated by the antibodies againstgoat estrogen receptors which lay the methodology and morphological basis for further studyon the biological function of goats’ estrogen receptor.