摘要
目的:构建含有GRO-α(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-α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-α原核表达质粒,高效表达GRO-α-GST融合蛋白,大小为34ku。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-α-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。
Objective：To construct a recombinant prokaryotic vector of GRO-α and optimize the condition for expressing GST fusion protein in E.coli BL21（DE3）,and discuss the function of GRO-α-GST fusion protein to promote proliferation of tumor cells. Methods： GRO-α 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-α-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-α vector has been successfully constructed. CCK-8 analysis showed that the fusion protein induced proliferation of MCF-7 cells. And GRO-α-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-α-GST protein may facilitate further research on its biological functions.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2010年第8期1060-1064,共5页
Journal of Nanjing Medical University(Natural Sciences)
基金
江苏省卫生厅资助课题(H200938)
