MBP-mCTCF融合蛋白原核表达载体的构建及表达

摘要

To construct the prokaryotic expression vector of pMal-C2G-mCTCF and induce the express~f fusion protein,DNA fragments encoding the mouse CTCF were generated by PCR using the plasmid pMXs-3Flag-mCTCF as a template,PCR products were digested with the restriction enzyme EcoR Ⅰ and SalⅠ and cloned into pMal-C2G expression vector.The plasmids were sequenced to confirm that there were no point mutations in the coding sequences.The constructed plasmids were transformed into E.coli BL21 to induce the expression of MBP-mCTCF fusion protein.The fusion protein was analyzed by SDS-PAGE and Coomassie blue staining.The prokaryotic expression plasmids pMal-C2G-mCTCF were successfully constructed,which were testified by bacterial PCR,gene sequence and double restriction enzyme digestion analysis.We also got the suitable conditions for inducing fusion protein expression.The optimized induction condition was 37 ℃ with 10 × 10-4 mol/L IPTG for 6 hours to induce higher amount of MBP-mCTCF fusion protein.%为了构建鼠的CTCF与麦芽糖结合蛋白(MBP)融合蛋白的原核表达载体,并进行原核诱导表达及鉴定,以pMXs-3Flag-mCTCF为模板,PCR扩增得到mCTCF序列,目的基因片段EcoR Ⅰ,SalⅠ双酶切后连接到同样双酶切的pMal-C2G载体上,测序鉴定.将测序正确的质粒转化到E.coli BL21中,用IPTG诱导融合蛋白的表达,用SDS-PAGE分离检测蛋白表达效果.经菌液PCR、测序鉴定、双酶切鉴定证明,得到的重组质粒pMal-C2G-mCTCF构建成功,且成功诱导出了MBP-mCTCF融合蛋白,最佳诱导条件为:10×10-4 mol/L IPTG浓度在37℃诱导6h.