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目的:构建含有Arf6-T27N基因的重组真核表达载体,并检测Arf6-T27N蛋白在MDA-MB-231人乳腺癌细胞中的表达情况,以期为深入研究Arf6在乳腺癌细胞迁移中的作用机制奠定基础。方法:从含Arf6-T27N基因的真核表达质粒pXS-Arf6-T27N中扩增出Arf6-T27N基因,插入pEGFP-N1载体中构建成pEGFP-N1/Arf6-T27N,利用脂质体将其转染MDA-MB-231人乳腺癌细胞。用新霉素筛选稳定表达的细胞系,Western blot确定重组蛋白表达,并用Boyden chamber小室实验检测转染Arf6-T27N的MDA-MB-231人乳腺癌细胞迁移能力的改变。结果:限制性内切酶鉴定和核酸定列测定证实成功构建了含Arf6-T27N的重组真核表达载体pEGFP-N1/Arf6-T27N。以重组质粒稳定转染MDA-MB-231人乳腺癌细胞,能检测到Arf6-T27N蛋白的表达,且Arf6-T27N蛋白能明显抑制MDA-MB-231人乳腺癌细胞的迁移。结论:成功构建了重组质粒pEGFP-N1/Arf6-T27N,稳定转染Arf6-T27N的MDA-MB-231人乳腺癌细胞可检测到其蛋白水平的稳定表达,重组质粒介导的Arf6-T27N蛋白能明显抑制MDA-MB-231细胞的迁移。
OBJECTIVE: To construct a recombinant eukaryotic expression vector containing Arf6-T27N gene and to detect the expression of Arf6-T27N protein in MDA-MB-231 human breast cancer cells in order to further investigate the mechanism of Arf6 in breast cancer cell migration Lay the foundation. Methods: Arf6-T27N gene was amplified from eukaryotic expression plasmid pXS-Arf6-T27N containing Arf6-T27N gene and inserted into pEGFP-N1 vector to construct pEGFP-N1 / Arf6-T27N. Dyeing MDA-MB-231 human breast cancer cells. Stably expressing cell lines were screened with neomycin, recombinant protein was identified by Western blot, and the migration ability of MDA-MB-231 human breast cancer cells transfected with Arf6-T27N was detected by Boyden chamber assay. Results: The restriction endonuclease assay and nucleic acid sequencing confirmed that the recombinant eukaryotic expression vector pEGFP-N1 / Arf6-T27N containing Arf6-T27N was successfully constructed. The recombinant plasmid was stably transfected into MDA-MB-231 human breast cancer cells, and the expression of Arf6-T27N protein was detected. Arf6-T27N protein could significantly inhibit the migration of MDA-MB-231 human breast cancer cells. CONCLUSION: The recombinant plasmid pEGFP-N1 / Arf6-T27N was successfully constructed and stable expression of Arf6-T27N protein was detected in MDA-MB-231 human breast cancer cells stably transfected with Arf6-T27N. Can significantly inhibit the migration of MDA-MB-231 cells.