GRO-?琢 的原核表达及对细胞增殖作用的初步研究
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江苏省卫生厅资助课题(H200938)


GRO-?琢 prokaryotic expression and the preliminary study of the effect on cell proliferation
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    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-

    Abstract:

    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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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(自然科学版),2010,(8):1060-1064

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  • 收稿日期:2010-02-01
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