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中华诊断学电子杂志 ›› 2015, Vol. 03 ›› Issue (01) : 1 -6. doi: 10.3877/cma.j.issn.2095-655X.2015.01.001

所属专题: 文献

肿瘤诊治研究

野生型和突变型乳腺癌标志基因C35蛋白的原核表达和纯化
尹昆1, 赵桂华1, 寇景轩1, 李琛琛1, 黄炳成1, 魏庆宽1, 闫歌1,()   
  1. 1. 272033 济宁,山东省医学科学院山东省寄生虫病防治研究所
  • 收稿日期:2014-08-02 出版日期:2015-02-26
  • 通信作者: 闫歌
  • 基金资助:
    国家自然科学基金(81041075); 山东省自然科学基金(Q2007D04)

Protein prokaryotic expression and purification of wild type and mutant C35 genes for breast carcinoma diagnosis

Kun Yin1, Guihua Zhao1, Jingxuan Kou1, Chenchen Li1, Bingcheng Huang1, Qingkuan Wei1, Ge Yan1,()   

  1. 1. Shandong Academy of Medical Science, Shandong Institute of Parasitical Disease, Jining 272033, China
  • Received:2014-08-02 Published:2015-02-26
  • Corresponding author: Ge Yan
  • About author:
    Corresponding author: Yan Ge, Email:
引用本文:

尹昆, 赵桂华, 寇景轩, 李琛琛, 黄炳成, 魏庆宽, 闫歌. 野生型和突变型乳腺癌标志基因C35蛋白的原核表达和纯化[J/OL]. 中华诊断学电子杂志, 2015, 03(01): 1-6.

Kun Yin, Guihua Zhao, Jingxuan Kou, Chenchen Li, Bingcheng Huang, Qingkuan Wei, Ge Yan. Protein prokaryotic expression and purification of wild type and mutant C35 genes for breast carcinoma diagnosis[J/OL]. Chinese Journal of Diagnostics(Electronic Edition), 2015, 03(01): 1-6.

目的

构建野生型和C112S突变型乳腺癌标志基因C35的原核表达载体,使其在原核细胞中高效表达、纯化并予以鉴定。

方法

聚合酶链反应(PCR)法扩增野生型和突变型C35基因,将其分别插入原核表达载体pGLO1中,构建重组融合蛋白表达载体野生型pGLO1-C35和突变型pGLO1-C35,转化大肠杆菌E.coli BL21,经异丙基硫代-β-D-半乳糖苷诱导表达C35融合蛋白,并对诱导条件进行优化,通过镍离子螯合亲和层析柱纯化野生型和突变型C35融合蛋白,经蛋白质印迹法验证纯化的重组蛋白特异性。

结果

成功构建了野生型和突变型C35基因的原核表达质粒,并将两种C35融合蛋白成功表达,最佳诱导表达条件为37℃,异丙基-β-D-硫代半乳糖苷诱导6 h,经纯化后获得高纯度的特异性重组蛋白,浓度达约1.5 mg/mL。

结论

在原核表达系统中成功表达、纯化了野生型和突变型C35融合蛋白,为进一步制备基于C35抗血清的乳腺癌早期筛查试剂盒奠定了基础。

Objective

To establish a prokaryotic expression system of the breast cancer marker gene C35 for breast carcinoma diagnosis.Wild type and C112S mutant of C35 prokaryotic recombinants were constructed and expressed the in E. coli strain BL21, and two kinds of C35 proteins were purified.

Methods

Sequences encoding wild type and C112S(open reading frame) of C35 were amplified by PCR using breast cancer cell line T47D cDNA, The PCR products were cloned into the BamHⅠand XhoI sites of the vector pGLO1.The positive recombinants of pGLO1-C35(wild type) and pGLO1-C35(C112S) were identified by double-digesting and sequencing, and were overexpressed in E. coli strain BL21, respectively.To optimize protein purification conditions, 10 mL of bacteria were incubated in lactose broth at 37℃ to an absorbance (A600) of 0.8, following different time gradients(3 h, 6 h and 9 h) with 1 mM isopropyl-D-1-thiogalactopyranoside(IPTG) at different temperature such as 37℃, 22℃ and 15℃.After the cells that carried C35 recombinants were induced by the optimized conditions and harvested, the generated bacteria were suspended in resuspension buffer and lysed by sonication, the supernatants were loaded onto the Ni2+ Chelating Sepharose Fast Flow column for affinity chromatography of the N-terminal 6×His tagged wild type or C112S C35 proteins.Finally, the wild type and C112S mutant of C35 proteins were identified by Western blot and quantified by ultramicro-spectrophotometer under 280 nm.

Results

The double-digesting and sequencing results indicated that both wild type and C112S mutant of C35 ORFs were successfully inserted into pGLO1 between BamHⅠand XhoI sites.Based on the time and temperature selections, the optimized conditions were identified as inducing with 1 mM IPTG for 4-6 h at 37℃, and high amount of C35 proteins could be expressed under these conditions.Both wild type and mutant C35 proteins were expressed soluble and chelated on Ni2+ sepharose beads with high affinity.Pure wild type and mutant C35 proteins were confirmed by SDS-PAGE and Western blot, indicating that the purified wild type and mutant C35 protein had high purity and fine immune activity.The final yield of purified wild type and mutant C35 proteins were about 1.5 mg/mL with a purity of about 90%.

Conclusions

The prokaryotic expression systems of wild type and mutant C35 gene are successfully established.The wild type and C112S mutant of C35 proteins with high purity and concentration could be yielded under the optimal expression conditions identified in this paper.Our work may be useful for developing breast carcinoma prophase diagnosis kit based on C35 antiserum.

图1 野生型和突变型C35基因的PCR扩增电泳图
图2 重组质粒pGLO1-C35和pGLO1-C35的双酶切鉴定图
图3 重组野生型和突变型C35蛋白的最佳诱导表达条件
图4 Ni柱大量纯化重组野生型和突变型C35蛋白的SDS-PAGE分析图
图5 纯化后的野生型和突变型C35蛋白水平比较
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