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。
Objective To construct the prokaryotic expression system of estrogen receptor α ligand bingding domain （hERα-LBD） and to evaluate the estrogen receptor ligand binding activity of the expressed protein. Methods hERα -LBD was amplicated from the plasmid of hERα -LBD by PCR, the identified PCR product was ligated with pGEM-T-easy vector to generate pGM-T-hERα -LBD. After the confirmation, the hERα -LBD fragments were obtained by enzyme digestion and inserted into pET-28a. The expression vectors were expressed in E.Coli to produce hERα-LBD protein. We mixed the hERα-LBD protein and estradiol and bovine serum albumin conjugated antigens （E2-BSA）, then evaluated the binding activity of hERα-LBD by electrophoresis. Results The amplified fragment was about 1.9 kb, which was in agreement with the expected target fragment. Recombinant plasmid of pGM-T-hERα -LBD was confirmed by enzyme digestion and sequencing, then pET-28a（＋）-hERα -LBD was constructed successfully. The expressed hERα-LBD protein in E.Coli was observed and the expression amount was 250 mg/L after affinity chromatography purification. hERα-LBD was confirmed to had estrogen binding activity by electrophoresis. Conclusion The prokaryotic expression system of pET-28a（＋）-hERα -LBD was successfully constructed, and hERα-LBD had the activity of binding.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2017年第1期61-65,共5页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(No.20507002)资助
