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目的:制备雌激素受体β多克隆抗体。方法:从人乳腺cDNA文库中PCR扩增ERβ AF1结构域(1~144位氨基酸),DNA重组插入原核表达质粒pGEX-KG,转化大肠杆菌DH5α,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达GST-ERβ AF1融合蛋白。纯化后免疫BALB/c小鼠,制备鼠多抗血清。通过Western blot和免疫组织化学实验鉴定血清特异性和效价。结果:成功构建了pGEX-KG/ERβ AF1原核表达载体,转化DH5α后可高效表达融合蛋白GST-ERβ AF1,免疫产生的ERβ多抗可特异检测ERβ真核表达载体转染人293T细胞及乳腺癌细胞中ERβ的表达。结论:制备的ER抗体对ERβ有反应原性,效价高,特异性好,为进一步研究ERβ的生物学功能奠定基础。
Objective: To prepare estrogen receptor β polyclonal antibody. METHODS: The ERβ AF1 domain (amino acids 1 to 144) was amplified by PCR from a human breast cDNA library. The recombinant plasmid was inserted into prokaryotic expression vector pGEX-KG and transformed into E. coli DH5α and isopropyl-β-D-thiogalactam Glycoside (IPTG) induces the expression of the GST-ERβ AF1 fusion protein. BALB / c mice were immunized after purification to prepare multi-antiserum. Serum specificity and titer were identified by Western blot and immunohistochemistry. Results: The prokaryotic expression vector pGEX-KG / ERβ AF1 was constructed successfully, and the fusion protein GST-ERβ AF1 was highly expressed after transformed into DH5α. The ERβ polyclonal antibody could specifically detect the ERβ eukaryotic expression vector and transfected into human 293T cells and breast cancer ERβ expression in cells. CONCLUSION: The prepared ER antibody has the reactivity with ERβ, high titer and good specificity, which lays the foundation for further study on the biological function of ERβ.