山羊β雌激素受体多克隆抗体的制备及免疫组化方法的建立

摘要

本研究旨在克隆济宁青山羊雌激素受体β(ERβ)基因,进行原核表达,制备多克隆抗体,建立免疫组化分析方法,检测济宁青山羊卵巢和子宫内ERβ分布.根据GenBank发布的绵羊ERβ基因序列设计一对引物,应用RT-PCR方法从济宁青山羊卵巢组织中扩增ERβ部分基因.经双酶切和测序分析后,连接到原核表达载体pET32a(+),构建重组表达载体pET32a(+)-ERβ,转化至大肠杆菌BL21 (DE3)中,IPTG诱导表达,表达产物经SDS-PAGE和Western blotting分析鉴定；经Ni-NTA纯化融合蛋白后免疫新西兰大白兔,制备多克隆抗体,并检测抗体效价及特异性；使用该抗体检测济宁青山羊卵巢和子宫组织细胞中的ERβ分布.结果表明,成功扩增出ERβ部分基因,并构建原核表达质粒,转化至大肠杆菌中表达出相对分子质量约为53 ku的融合蛋白；Western blotting证明该融合蛋白能与兔抗人的ERβ多克隆抗体特异性反应.制备的多克隆抗体经ELISA检测效价达到1∶213,以此抗体代替购置的标准兔抗人ERβ抗体,进行Western blotting反应,特异性良好；使用该抗体建立免疫组化方法检测结果表明济宁青山羊卵巢颗粒细胞和子宫内膜上皮细胞、平滑肌细胞存在ERβ的分布.本研究获得特异性兔抗山羊ERβ多克隆抗体,使用该抗体建立免疫组化方法首次报道了济宁青山羊卵巢和子宫ERβ的分布表达,为进一步研究β雌激素受体的生物学功能奠定了方法学和形态学基础.%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 o-vary 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 BL2( 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 ERfi 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.