南昌大学学报（医学版）
南昌大學學報（醫學版）
남창대학학보（의학판）
ACTA ACADEMIAE MEDICINAE JIANGXI
2015年
1期
1-3,14
,共4页
CTCF%原核表达载体%谷胱甘肽转移酶%重组蛋白表达
CTCF%原覈錶達載體%穀胱甘肽轉移酶%重組蛋白錶達
CTCF%원핵표체재체%곡광감태전이매%중조단백표체
CCCTC-binding factor%prokaryotic expression vector%glutathione S-transferase%recombinant protein expression
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。
目的：構建人 CCCTC 結閤因子（CCCTC-binding factor，CTCF）與穀胱甘肽轉移酶（glutathione S-transfer-ase，GST）重組蛋白的原覈錶達載體，併進行誘導錶達。方法以 pEASY-T1-SIMPLE-CTCF 為模闆，設計引入限製性酶切位點的 CTCF 引物，PCR 方法擴增目的片段，產物經限製性內切酶酶切後與原覈錶達載體 pGEX-4T-1連接，構建成 pGEX-4T-1-CTCF 原覈錶達載體，轉化至大腸埃希菌 BL21中，經異丙基硫代-β-D 半乳糖苷（IPTG）進行誘導，Western blot 檢測 GST-CTCF 融閤蛋白的錶達情況。結果經菌液 PCR、質粒雙酶切分析、基因測序分析證實重組錶達質粒 pGEX-4T-1-CTCF 構建成功，GST-CTCF 融閤蛋白產物經 Western blot 鑒定為特異性錶達。結論成功構建瞭 pGEX-4T-1-CTCF 原覈錶達質粒，穫得特異性的 GST-CTCF 融閤蛋白，為進一步研究轉錄因子CTCF 的功能奠定瞭基礎。
목적：구건인 CCCTC 결합인자（CCCTC-binding factor，CTCF）여곡광감태전이매（glutathione S-transfer-ase，GST）중조단백적원핵표체재체，병진행유도표체。방법이 pEASY-T1-SIMPLE-CTCF 위모판，설계인입한제성매절위점적 CTCF 인물，PCR 방법확증목적편단，산물경한제성내절매매절후여원핵표체재체 pGEX-4T-1련접，구건성 pGEX-4T-1-CTCF 원핵표체재체，전화지대장애희균 BL21중，경이병기류대-β-D 반유당감（IPTG）진행유도，Western blot 검측 GST-CTCF 융합단백적표체정황。결과경균액 PCR、질립쌍매절분석、기인측서분석증실중조표체질립 pGEX-4T-1-CTCF 구건성공，GST-CTCF 융합단백산물경 Western blot 감정위특이성표체。결론성공구건료 pGEX-4T-1-CTCF 원핵표체질립，획득특이성적 GST-CTCF 융합단백，위진일보연구전록인자CTCF 적공능전정료기출。
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.
