构建STK11基因融合质粒检测其对肺癌细胞迁移能力的影响

Construction of STK11 Gene Fusion Plasmid and Its Effect on Migration of Lung Cancer Cells

目的构建表达STK11(serine/threonine kinase 11,STK11)基因和报告基因EGFP融合蛋白的重组质粒,并转染肺癌A549和H460细胞株,检测其对肺癌细胞迁移能力的影响。方法构建重组质粒pEGFP-STK11,双酶切和测序进行鉴定。重组质粒转染A549和H460细胞株,荧光显微镜观察EGFP蛋白表达情况,Western blot检测STK11蛋白表达情况,划痕实验检测其对肺癌细胞迁移能力的影响。结果酶切和测序结果表明pEGFP-STK11重组质粒构建成功;在转染A549和H460细胞后24h观察到绿色荧光最强;Western blot结果显示STK11蛋白在转染后24 h条带最深;划痕实验显示,转染组细胞迁移距离小于0.9%氯化钠溶液对照组(P<0.05)。结论成功构建STK11基因重组质粒,并能转染A549和H460细胞,转染后对肺癌细胞迁移能力起一定抑制作用。

Objective To construct eukaryotic expression plasmids containing enhanced green fluorescent protein(EGFP) gene and serine/threonine kinase 11(STKK11) gene, and to explore its effect on migration of A549 and H460 cells. Methods Fusion plasmid of pEGFP-STK11 was constructed and identified by double restriction enzyme digestion and gene sequencing. The resultant plasmid was transfected into A549 and H460 cells respectively. The expression of EGFP and STK11 were observed by fluorescence microscopy and Western blot analysis. The cell migration ability was tested by Wound-healing assay. Results Enzyme digestion and DNA sequence analysis showed the recombinant vector of pEGFP-STK11 was constructed successfully. Fluorescence microscopy and Western blot analysis indicated STK11 gene was overexpressed in A549 and H460 cells after transfection, and reached the peak after 24 h. Wound-healing assay revealed cell migration distance of STK11 gene transfected group was significant less than that of control group(P<0.05). Conclusion STK11 gene fusion plasmid is constructed and transfected into lung cancer cell lines A549 and H460 successfully. Cells migration is inhibited to some extent after transfection.