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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 expression vector containing GRO-α (growth regulatory oncogene-1), and to optimize the expression of recombinant protein in E. coli BL21 (DE3) and analyze its effect on the proliferation of tumor cells. Methods: The gene fragment of GRO-α was amplified by RT-PCR, cloned into prokaryotic expression vector pGEX-4T-1 and induced by IPTG. The expression of target protein was detected by SDS-PAGE and Western blot. Purification of the target protein by affinity chromatography, the role of human breast cancer cell line MCF-7, the application of CCK8 detection of cell proliferation. Results: The prokaryotic expression plasmid pGEX-4T-1 / GRO-α was successfully constructed and the expression level of GRO-α-GST fusion protein was 34ku. The results of cell proliferation assay showed that this protein could promote the proliferation of human breast cancer cell MCF-7. CONCLUSION: Recombinant recombinant GRO-α-GST fusion protein has the biological activity to promote the proliferation of human breast cancer cells and lay a foundation for the future preparation of corresponding antibodies and provide a new method for targeted therapy of breast cancer.