摘要
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p GLO1中,构建重组融合蛋白表达载体野生型p GLO1-C35和突变型p GLO1-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/m L。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 C 112S 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 BamH I and XhoI sites of the vector pGLO 1.The positive recombinants of pGLO 1-C35 （ wild type ） and pGLO 1-C35 （ C 112S） 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~C 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~C , 22~C and 15~C. 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 superuatants were loaded onto the Ni2＋ Chelating Sepharose Fast Flow column for affinity chromatography of the N-terminal 6xHis 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 ultranficro- spectrophotometer under 280 nm.Results The double-digesting and sequencing results indicated that both wild type and Cl12S mutant of C35 ORFs were successfully inserted into pGLO1 between BamH I and 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 Nia＋ sepharose beads with high affinity. Pure wild type and mutant C35 proteins were confirmed by SDS-PAGE and Westem 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 Cl12S 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.
出处
《中华诊断学电子杂志》
2015年第1期1-5,共5页
Chinese Journal of Diagnostics(Electronic Edition)
基金
国家自然科学基金(81041075)
山东省自然科学基金(Q2007D04)
关键词
C35基因
基因
肿瘤
原核表达纯化
乳腺肿瘤
诊断
C35 gene
Genes, neoplasm
Prokaryotic expression and purification
Breastreoplasms
Diagnosis
