[ Objective] To express and purify the intracellular hydrophilic domains of bovine membrane carrier proteins：anion exchanger, member 1 （AE1） and electregenic sodium bicarbonate cotransporter 1 （NBCel）, which were...[ Objective] To express and purify the intracellular hydrophilic domains of bovine membrane carrier proteins：anion exchanger, member 1 （AE1） and electregenic sodium bicarbonate cotransporter 1 （NBCel）, which were associated with bicarbonate ion transport. [ Method] The hydrophilic domains of bovine AE1 and NBCel were amplified by PCR and inserted into the prokaryotic expression vector pET-28a, respectively. The recombinant plasmids were transformed into the expression strain E. coli BL21 （DE3） and then induced by IPTG. The expressed proteins were purified by nickel ion affinity chromatography and analyzed by 15% SDS-PAGE. [Result] The hydrophilic domains of bovine AE1 and NBCel were amplified respectively by PCR and expressed by prokaryotic expression system with the induction of IPTG. They were mainly expressed in the cyto- plasm of E. coli and high-purity was achieved by nickel ion affinity chromatography. [Condusion] The expression of the hydrophilic domains of bovine AE1 and NBCel provides a major exit route for preparation of antibodies and the regulatory mechanisms of carrier proteins.展开更多
胃癌位居我国恶性肿瘤死因的第二位。95%以上的胃癌为腺癌,对放疗和化疗都不敏感;已发生转移的胃癌5年生存率仅15%～20%。因此,寻求有效的早期诊断和靶向治疗方法一直是胃癌研究关注的焦点。该课题组在对人红细胞阴离子交换蛋白1(AE1)...胃癌位居我国恶性肿瘤死因的第二位。95%以上的胃癌为腺癌,对放疗和化疗都不敏感;已发生转移的胃癌5年生存率仅15%～20%。因此,寻求有效的早期诊断和靶向治疗方法一直是胃癌研究关注的焦点。该课题组在对人红细胞阴离子交换蛋白1(AE1)的研究中发现,AE1除特异表达在红细胞膜外,在胃癌细胞质中具有83%的高频率表达。AE1的异常表达与碱性环境下p65/miR-24负反馈调控失衡相关。胃上皮细胞内缺乏AE1上膜途径导致AE1表达后大量滞留于细胞质中,胞质内的AE1通过几个相互关联的信号通路参与肿瘤的发生发展进程,主要包括:①与肿瘤抑制蛋白p16直接相互作用,扣押后者在胞质而不能入核发挥细胞周期负调控作用;②妨碍另一家族成员AE2正常上膜,加速AE2降解,进一步促进细胞碱化和Wnt/β-catenin通路活化。这一研究成果已被收录在Atlas of Genetics and Cytogenetics in Oncology and Haematology中。明确了AE1是胃癌分子标志物,在此基础上,课题组在细胞及动物水平开展了针对AE1的胃癌靶向治疗研究。结果显示,靶向AE1可以有效抑制胃癌细胞增殖,能够使药物诱发的小鼠胃癌检出率由62%～70%降低到15.8%。AE1蛋白作为胃癌诊断和治疗的靶标具有非常重要的应用价值。展开更多
利用 PCR方法 ,从阴离子交换蛋白 1(AE1)全长 c DNA中扩增出约 35 0 bp c末端 c DNA片段。测序后将其克隆至 p GADT7载体上。用醋酸锂法将构建好的 p GADT7- AE1- c-末端转染酵母菌 AH10 9,观察其在选择性培养基上的表达情况。结果表明 ...利用 PCR方法 ,从阴离子交换蛋白 1(AE1)全长 c DNA中扩增出约 35 0 bp c末端 c DNA片段。测序后将其克隆至 p GADT7载体上。用醋酸锂法将构建好的 p GADT7- AE1- c-末端转染酵母菌 AH10 9,观察其在选择性培养基上的表达情况。结果表明 ,获得了 35 0 bp AE1c-末端 c DNA,p GADT7- AE1- c-末端对酵母无毒性 ,不能激活检测基因 。展开更多
基金financially supported by Key Project of Jiangsu Science and Technology Department (BC2004365)
文摘[ Objective] To express and purify the intracellular hydrophilic domains of bovine membrane carrier proteins：anion exchanger, member 1 （AE1） and electregenic sodium bicarbonate cotransporter 1 （NBCel）, which were associated with bicarbonate ion transport. [ Method] The hydrophilic domains of bovine AE1 and NBCel were amplified by PCR and inserted into the prokaryotic expression vector pET-28a, respectively. The recombinant plasmids were transformed into the expression strain E. coli BL21 （DE3） and then induced by IPTG. The expressed proteins were purified by nickel ion affinity chromatography and analyzed by 15% SDS-PAGE. [Result] The hydrophilic domains of bovine AE1 and NBCel were amplified respectively by PCR and expressed by prokaryotic expression system with the induction of IPTG. They were mainly expressed in the cyto- plasm of E. coli and high-purity was achieved by nickel ion affinity chromatography. [Condusion] The expression of the hydrophilic domains of bovine AE1 and NBCel provides a major exit route for preparation of antibodies and the regulatory mechanisms of carrier proteins.
文摘胃癌位居我国恶性肿瘤死因的第二位。95%以上的胃癌为腺癌,对放疗和化疗都不敏感;已发生转移的胃癌5年生存率仅15%～20%。因此,寻求有效的早期诊断和靶向治疗方法一直是胃癌研究关注的焦点。该课题组在对人红细胞阴离子交换蛋白1(AE1)的研究中发现,AE1除特异表达在红细胞膜外,在胃癌细胞质中具有83%的高频率表达。AE1的异常表达与碱性环境下p65/miR-24负反馈调控失衡相关。胃上皮细胞内缺乏AE1上膜途径导致AE1表达后大量滞留于细胞质中,胞质内的AE1通过几个相互关联的信号通路参与肿瘤的发生发展进程,主要包括:①与肿瘤抑制蛋白p16直接相互作用,扣押后者在胞质而不能入核发挥细胞周期负调控作用;②妨碍另一家族成员AE2正常上膜,加速AE2降解,进一步促进细胞碱化和Wnt/β-catenin通路活化。这一研究成果已被收录在Atlas of Genetics and Cytogenetics in Oncology and Haematology中。明确了AE1是胃癌分子标志物,在此基础上,课题组在细胞及动物水平开展了针对AE1的胃癌靶向治疗研究。结果显示,靶向AE1可以有效抑制胃癌细胞增殖,能够使药物诱发的小鼠胃癌检出率由62%～70%降低到15.8%。AE1蛋白作为胃癌诊断和治疗的靶标具有非常重要的应用价值。
文摘利用 PCR方法 ,从阴离子交换蛋白 1(AE1)全长 c DNA中扩增出约 35 0 bp c末端 c DNA片段。测序后将其克隆至 p GADT7载体上。用醋酸锂法将构建好的 p GADT7- AE1- c-末端转染酵母菌 AH10 9,观察其在选择性培养基上的表达情况。结果表明 ,获得了 35 0 bp AE1c-末端 c DNA,p GADT7- AE1- c-末端对酵母无毒性 ,不能激活检测基因 。
