文章摘要
陈晓笑,唐〓奇,朱 进,仇镇宁,李玉华,王祝鸣,管晓虹,冯振卿.GRO-?琢 的原核表达及对细胞增殖作用的初步研究[J].南京医科大学学报,2010,(8):1060~1064
GRO-?琢 的原核表达及对细胞增殖作用的初步研究
GRO-?琢 prokaryotic expression and the preliminary study of the effect on cell proliferation
投稿时间:2010-02-01  
DOI:10.7655
中文关键词: GRO-ɑ  重组蛋白  CCK8  MCF-7
英文关键词: GRO-?琢  recombinant protein  CCK8  MCF-7
基金项目:江苏省卫生厅资助课题(H200938)
作者单位
陈晓笑 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京〓210029 
唐〓奇 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京〓210029 
朱 进 南京军区军事医学研究所,江苏 南京〓210002 
仇镇宁 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京〓210029 
李玉华 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京〓210029 
王祝鸣 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京〓210029 
管晓虹 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京〓210029 
冯振卿 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京〓210029 
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中文摘要:
      目的:构建含有GRO-?琢(生长调节癌基因-1)的重组表达载体,优化重组蛋白在E.coli BL21(DE3)中表达,分析其对肿瘤细胞的增殖作用的影响?方法:RT-PCR扩增GRO-?琢基因片段,克隆于原核表达载体pGEX-4T-1,IPTG进行诱导表达,SDS-PAGE及Western blot检测目的蛋白表达?经亲和层析柱纯化目的蛋白,作用于人乳腺癌细胞系MCF-7,应用CCK8检测其对细胞增殖的影响?结果:成功构建pGEX-4T-1/GRO-?琢原核表达质粒,高效表达GRO-?琢-GST融合蛋白,大小为34 ku?细胞增殖实验结果显示,该蛋白对人乳腺癌细胞MCF-7增殖具有促进作用?结论:重组GRO-?琢-GST融合蛋白具有生物学活性,可促进人乳腺癌细胞的增殖,为今后制备相应抗体奠定基础并为乳腺癌的靶向治疗提供新的方法?
英文摘要:
      Objective:To construct a recombinant prokaryotic vector of GRO-?琢 and optimize the condition for expressing GST fusion protein in E.coli BL21(DE3),and discuss the function of GRO-?琢-GST fusion protein to promote proliferation of tumor cells. Methods:GRO-?琢 gene was amplified by RT-PCR method and inserted into the prokaryotic express vector pGEX-4T-1. The plasmid was transformed into E.coli BL21(DE3) and induced to express fusion protein GRO-?琢-GST with IPTG. The fusion protein was detected using SDS-PAGE and Western-blot methods. The fusion protein was purified by GSTrap affinity column,and its function on tumor cells proliferation was detected by CCK-8 method in MCF-7 cells. Results:Recombinant pGEX-4T-1/GRO-?琢 vector has been successfully constructed. CCK-8 analysis showed that the fusion protein induced proliferation of MCF-7 cells. And GRO-?琢-GST fusion protein with high purity was obtained. Conclusion:The protein has a biological activity to induce proliferation of MCF-7 cells,and the study of GRO-?琢-GST protein may facilitate further research on its biological functions.
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