hERα-LBD原核表达载体的构建及蛋白表达活性鉴定

发布时间：2019-08-05 10:34

【摘要】：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。
[Abstract]:Objective to construct the prokaryotic expression vector of human estrogen receptor 伪 subunit ligand binding domain (hER 伪-LBD) and identify the activity of the expressed protein. Methods the hER 伪-LBD gene was amplified by PCR using hER 伪-LBD plasmid as template. The PCR amplification product was identified and ligated with T vector (pGEM-T-easy). The ligator was transformed into receptive cells. The positive colony was identified by restriction enzyme digestion and sequencing, and the pET-28a () plasmid was double digested and ligated by T4DNA ligase respectively. The recombinant expression vector pET-28a ()-hER 伪-LBD, was constructed. Transformed into E. coli JM109 and BL21 receptive cells. Isopropyl-尾-D-galactoside (IPTG) induced the expression of hER 伪-LBD fusion protein in cells, and the estrogen ligand binding activity of hER 伪-LBD was identified by electrophoresis by mixing E2-bovine serum albumin (BSA) coupling antigen (E2-BSA) with fusion protein. Results the hER 伪-LBD gene fragment amplified by PCR was about 1.9 KB, which was consistent with the expected target fragment size. The recombinant plasmid pGM-T-hER 伪-LBD was formed and transformed into receptive cells. The positive bacteria were identified correctly by restriction endonuclease digestion and sequencing, and ligated with pET-28a () plasmid. The recombinant prokaryotic expression vector pET-28a ()-hER 伪-LBD; was successfully constructed and the ligand binding region hER 伪-LBD protein of human estrogen receptor ER 伪 was heterogeneously expressed in E. coli. After purification by affinity chromatography, the expression of hER 伪-LBD protein could reach 250 mg 路L ~ (- 1). It was confirmed by electrophoresis that hER 伪-LBD had estrogenic binding activity. Conclusion the recombinant prokaryotic expression vector pET-28a ()-hER 伪-LBD,hER 伪-LBD has estrogen binding activity.
【作者单位】：包头医学院基础医学与法医学院;包头医学院第一附属医院;河北北方学院;北京科技大学;
【基金】：国家自然科学基金(No.20507002)资助
【分类号】：R114

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