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多功能转录因子CTCF重组质粒构建及其表达鉴定 被引量：2

Cloning,Expression,Purification and Identification of Multi-potent Transcription Factor CTCF Recombinant and Its Segments
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摘要 目的构建人CTCFcDNA全长及N端、Zn指、C端3个片段的原核融合表达载体,纯化融合蛋白并进行鉴定。方法以编码CTCF全长序列的质粒为PCR模板,分别构建GST融合表达重组质粒pGEX-4T-2-CTCF,pGEX-4T-2-CTCF-N,pGEX-4T-2-CTCF-Zn,pGEX-4T-2-CTCF-C,转化大肠杆菌BL21,酶切、测序鉴定;优化IPTG诱导表达条件,并对亲和层析纯化的GST融合蛋白CTCF,CTCF-N,CTCF-Zn,CTCF进行Far-Western blot鉴定。结果重组质粒经双酶切和测序鉴定证实构建成功。GST融合蛋白CTCF,CTCF-N,CTCF-Zn,CTCF-C均在大肠杆菌中成功诱导表达,融合蛋白经亲和层析纯化获得纯化蛋白。各纯化蛋白经SDS-PAGE和Far-Western blot鉴定,均为诱导表达蛋白质。结论成功构建了GST融合表达CTCF,CTCF-N,CTCF-Zn,CTCF-C的重组质粒,对融合蛋白表达条件进行了优化,获得了高效表达的GST融合蛋白。 Objective To clone ctcf eDNA, N, Zn and C fragments separately into expresstion vector, purify and identify the expressed proteins. Methods Using the recombinant plasmid pGEM7Zf （-）-ctcf as template for PCR, pGEX-4T-2-ctcf, pGEX-4T-2-ctcf-N, pGEX-4T-2- ctcf-Zn and pGEX-4T-2- ctcf-C recombinants were constructed successfully. After transformed into E. coli BL21 cells, the recombinants were confirmed by enzyme digestion and sequence analysis. After optimizing the IPTG inducing condition, the purified GST fusion proteins with affinity chromatography were conformed by Far-Western blotting. Results The recombinant plasmids pGEX-4T-2- ctcf, pGEX-4T-2- ctcf-N, pGEX-4T-2-ctcf-Zn and pGEX-4T-2-ctcf-C were confirmed by restriction enzyme assay and sequencing. All GST fusion proteins, CTCF, CTCF-N, CTCF-Zn and CTCF-C were successfully expressed at the optimal parameters and purified with affinity chromatography, and specifically recognized by anti-GST antibody. Conclusion Ctcf, ctcf-n, ctcf-Zn and ctcf-c gene recombinants were constructed successfully and their corresponding fusion proteins were successfully purified with affinity chromatography and identified.
作者 蒋磊 覃扬 孙芝琳 武静 杨文理 张军哲
机构地区 四川大学华西基础医学与法医学院生物化学与分子生物学教研室
出处 《四川大学学报（医学版）》 CAS CSCD 北大核心 2009年第1期1-5,共5页 Journal of Sichuan University(Medical Sciences)
基金 国家自然科学基金(批准号30470792)资助
关键词 CTCF结合因子 融合蛋白 表达 CTCF binding factor Fusion protein Expression
分类号 Q78 [生物学—分子生物学]
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参考文献16

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  • 2Klenova EM, Nicolas RH, Paterson HF, et al. CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms. Mol Cell Biol, 1993,13(12):7612-7624.
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同被引文献27

  • 1何莉,朱旭东,毛志勇,朱恒奇,黄培堂.转录因子CTCF的克隆、表达及其抗体制备[J].生物技术通讯,2004,15(5):454-456. 被引量：5
  • 2YEH A , WEI M , GOLUB S B , et al . Chromosome arm 16q in Wilms tumors: unbalanced chromosomal translocations, loss of heterozygosity, and assessment of the CTCF gene[J]. Genes Chromosomes. Cancer, 2002, 35 (2): 156- 163.
  • 3FILIPPOVA G N , LINDBLOM A , MEINCKE L J , et al.A widely expressed transcription factor with multiple DNA sequence specificity, CTCF, is localized at chromosome segment 16q22. 1 within one of the smallest regions of overlap for common deletions in breast and prostate cancers[J]. Genes Chromosomea. Cancer, 1998, 22(1): 26-36.
  • 4DARCI T , BUTCHERA D I , RODENHISER. Epigenetic inactivation of BRCA1 is associated with aberrant expression of CTCF and DNA methyltransferase (DNMT3B) in some sporadic breast tumours[J]. European Journal of Cancer, 2007, 43(1): 210-219.
  • 5DOCQUIER F , FARRAR D, D'ARCY V , et at . Heightened expression of CTCF in breast cancer cells is associated with resistance to apoptosis[J]. Cancer Res, 2005, 65(12): 5112-5122.
  • 6KLENOVA E M, NICOLAS R H, CARBE A F, et al. Molecular weight abnormalities of the CTCF transcription factor : CTCF migrates aberrantly in SDS-PAGE and the size of the expressed protein is affected by the UTEs and sequences within the coding region of the CTCF gene[J]. Nucleic Acids Res, 1997, 25(3): 466-474.
  • 7LOUKINOV D I , PUGACHEVA E , VATOLIN S , et al. BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11- zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma[J]. Proc Natl Acad Sci USA, 2002, 99(10): 6806-6811.
  • 8VOSTROV A A , TAHENY M J , QUITSCHKE W W. A region to the N-terminal side of the CTCF zinc finger domain is essential for activating transcription from the amyloid precursor protein[J]. J Biol Chem, 2002, 277(2): 1619-1627.
  • 9OHLSSON R , RENKAWITZ R , LOBANENKOV V. CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease[J]. Trends Genet, 2001, 17 (9) :520-527.
  • 10RASKO J E , KLENOVA E M , LEON J, et al . Cell growth inhibition by the multifunctional multivalent zinc-finger factor CTCF[J]. Cancer Res, 2001, 61(16): 6002-6007.

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四川大学学报（医学版）
2009年 第1期

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