人抑癌基因NKX2-3真核表达载体的构建及其对肿瘤细胞生长的作用

Construction of Flag-Tagged Human NKX2-3 Eukaryotic Expression Vector and its Role on Tumor Cell Growth

目的:构建人抑癌基因NKX2-3的真核表达载体,并验证其对肿瘤细胞生长的影响。方法:采用PCR技术从人乳腺文库中扩增人NKX2-3基因,将其克隆到Flag-pc DNA3.0载体中,酶切和测序验证后转染人胚肾293T细胞,蛋白免疫印迹鉴定其表达;转染人结肠癌细胞HCT-116和肝癌Hep G2细胞,通过CCK8法测定细胞生长曲线。结果:从人乳腺文库中扩增得到约1080 bp的DNA片段,并克隆至Flag-pc DNA3.0载体上,测序结果与目的序列完全一致;转染人胚肾293T细胞后,目的基因获得表达;细胞生长曲线结果显示,转染Flag-NKX2-3的人结肠癌、肝癌细胞较空载体细胞生长慢。结论:构建了Flag-NKX2-3真核表达载体,为进一步研究NKX2-3在肿瘤中的功能奠定了基础。

Objective：To construct the eukaryotic expression vector of human NKX2-3 gene labeled with Flagtag and detect its role on tumor cell growth.Methods：Human NKX2-3 coding region was amplified from humanmammary c DNA library by PCR and cloned into Flag-pc DNA3.0 vector.The recombinant plasmid Flag-NKX2-3was identified by enzyme digestion and sequencing,transfected into colorectal cancer and hepatic cancer cell lineand detected by Western blot.Then CCK8 assay was examined to investigate Flag-NKX2-3 transfected cell prolif-eration.The proliferation in HCT-116 and Hep G2 cells which were supposed to be inhibited by Flag-NKX2-3was identified by its expression.Results：The eukaryotic expression vector of human NKX2-3 labeled with Flagtag was successfully constructed by double digestion identification.The inserted fragment was verified to be correctby sequencing.Human NKX2.3 protein was expressed in human HCT-116 and Hep G2 cells shown by Westernblot.Cell growth curve showed that cells transfectd with Flag-NKX2-3 grew significantly slower than the controlcells.Conclusion：The eukaryotic expression vector of Flag-NKX2-3 was successfully constructed which lays a mo-lecular foundation for the research of NKX2-3 in cancer development and progression.