GST-CTCF 融合蛋白原核表达载体的构建及表达

摘要

目的：构建人 CCCTC 结合因子（CCCTC-binding factor，CTCF）与谷胱甘肽转移酶（glutathione S-transfer-ase，GST）重组蛋白的原核表达载体，并进行诱导表达。方法以 pEASY-T1-SIMPLE-CTCF 为模板，设计引入限制性酶切位点的 CTCF 引物，PCR 方法扩增目的片段，产物经限制性内切酶酶切后与原核表达载体 pGEX-4T-1连接，构建成 pGEX-4T-1-CTCF 原核表达载体，转化至大肠埃希菌 BL21中，经异丙基硫代-β-D 半乳糖苷（IPTG）进行诱导，Western blot 检测 GST-CTCF 融合蛋白的表达情况。结果经菌液 PCR、质粒双酶切分析、基因测序分析证实重组表达质粒 pGEX-4T-1-CTCF 构建成功，GST-CTCF 融合蛋白产物经 Western blot 鉴定为特异性表达。结论成功构建了 pGEX-4T-1-CTCF 原核表达质粒，获得特异性的 GST-CTCF 融合蛋白，为进一步研究转录因子CTCF 的功能奠定了基础。%Objective To construct the prokaryotic expression vector for inducing the expres-sion of recombinant human CCCTC-binding factor(CTCF)-glutathione S-transferase(GST)fusion protein.Methods The primers with BamHⅠand Not I endonuclease sites were designed and the target gene encoding CTCF full-length protein was amplified by PCR with pEASY-T1-SIMPLE-CTCF as the template.After digestion with restriction endonuclease,the CTCF target fragment was inserted into pGEX-4T-1 to create the prokaryotic expression vector pGEX-4T-1-CTCF, which was transformed into E.coli BL21 and then was induced by isopropyl-β-D-thiogalactopyr-anoside.The expression of GST-CTCF fusion protein was detected by Western blot.Results The prokaryotic expression vector pGEX-4T-1-CTCF was successfully constructed,which was testi-fied by PCR detection of bacterial liquid,double restriction enzyme digestion analysis and gene se-quencing.Western blot analysis revealed that the GST-CTCF fusion protein was specifically ex-pressed after IPTG induction.Conclusion The prokaryotic expression vector pGEX-4T-1-CTCF was successfully constructed and the specific GST-CTCF fusion protein was obtained.Our results laid a foundation for the study of function of transcription factor CTCF.