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目的构建人雌激素受体α亚型配体结合区(hERα-LBD)的原核表达载体,并进行表达蛋白的活性鉴定。方法以hERα-LBD质粒为模板,采用PCR方法扩增hERα-LBD基因,PCR扩增产物鉴定后与T载体(pGEM-T-easy)连接,将连接物转化到感受态细胞中,阳性菌落经酶切和测序鉴定,和pET-28a(+)质粒分别进行双酶切,T4DNA连接酶连接,构建重组表达载体pET-28a(+)-hERα-LBD,转化到大肠杆菌JM109和BL21感受态细胞中。异丙基-β-D-硫代半乳糖苷(IPTG)诱导细胞表达hERα-LBD融合蛋白;将雌二醇-牛血清白蛋白(BSA)偶联抗原(E2-BSA)与融合蛋白混合,通过电泳法鉴定hERα-LBD的雌激素配体结合活性。结果 PCR扩增的hERα-LBD基因片段约为1.9kb,与预期目的片段大小一致。与T载体连接,形成pGM-T-hERα-LBD重组质粒,转化到感受态细胞,阳性菌落酶切和测序鉴定正确,与pET-28a(+)质粒酶切后连接,成功构建原核重组表达载体pET-28a(+)-hERα-LBD;在大肠杆菌中异源表达人雌激素受体ERα的配体结合区hERα-LBD蛋白。经亲和色谱法纯化后,hERα-LBD蛋白的表达量可达250mg/L。电泳法证实hERα-LBD具有雌激素结合活性。结论成功构建原核重组表达载体pET-28a(+)-hERα-LBD,hERα-LBD具有雌激素结合活性。
Objective To construct a prokaryotic expression vector of human estrogen receptor α subtype ligand binding region (hERα-LBD), and to identify the expression of the expressed protein. Methods hERα-LBD plasmid was used as a template to amplify hERα-LBD gene by PCR. The PCR product was identified by PCR and ligated with T vector (pGEM-T-easy). The conjugate was transformed into competent cells. The positive colonies The recombinant plasmid pET-28a (+) - hERα-LBD was transformed into E. coli JM109 and BL21 competent cells by digestion with T4 DNA ligase and digested with restriction endonucleases and sequencing. in. BSA-conjugated antigen (E2-BSA) was mixed with the fusion protein by using isopropyl-β-D-thiogalactopyranoside (IPTG) Estrogen ligand binding activity of hERα-LBD was identified by electrophoresis. Results The fragment of hERα-LBD gene amplified by PCR was about 1.9 kb, which was consistent with the expected fragment size. The recombinant plasmids pGM-T-hERα-LBD were transformed into competent cells. The positive colonies were digested with restriction endonucleases and identified by sequencing. After digested with pET-28a (+) plasmid, the prokaryotic recombinant expression vector pET-28a (+) - hERα-LBD; ligand-binding region hERα-LBD protein heterologously expressed in human Estrogen receptor ERα in E. coli. Purified by affinity chromatography, the expression of hERα-LBD protein up to 250mg / L. Electrophoresis confirmed that hERα-LBD has estrogen-binding activity. Conclusion The prokaryotic recombinant expression vector pET-28a (+) - hERα-LBD was constructed successfully. The hERα-LBD has estrogen-binding activity.