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目的构建携带绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)标签和hNK4基因的重组腺病毒Ad5-hNK4-EGFP,并检测其对乳腺癌MDA-MB-231细胞增殖的影响。方法以pcDNA3/hNK4质粒为模板,采用高保真DNA聚合酶扩增hNK4基因,克隆至腺病毒穿梭载体pAdTrack-CMV中,构建重组穿梭质粒pAdTrack-hNK4,穿梭质粒经PmeⅠ酶切线性化,与腺病毒骨架质粒pAdEasy-1在大肠杆菌BJ5183中进行同源重组,获得重组腺病毒质粒pAd5-hNK4-EGFP,经PacⅠ酶切线性化后转染AD-293细胞进行包装,获得重组腺病毒Ad5-hNK4-EGFP,经4轮扩增后,测定病毒滴度;将重组腺病毒感染AD-293细胞,采用RT-PCR及Western blot法检测感染细胞中NK4基因的转录及蛋白的表达;MTT法检测Ad5-hNK4-EGFP对肝细胞生长因子(Hepatocyte growth factor,HGF)诱导的MDA-MB-231细胞增殖的影响。结果重组腺病毒质粒pAd5-hNK4-EGFP经PacⅠ酶切鉴定证明构建正确;经包装及4轮扩增后,重组腺病毒Ad5-hNK4-EGFP的滴度可达1.5×1011PFU/ml;经RT-PCR及Western blot鉴定表明,重组腺病毒携带的NK4基因能够在AD-293细胞中表达;HGF可促进MDA-MB-231细胞增殖,而Ad5-hNK4-EGFP可拮抗HGF诱导的MDA-MB-231细胞增殖。结论成功构建了表达hNK4基因的重组腺病毒Ad5-hNK4-EGFP,其可拮抗HGF诱导的MDA-MB-231细胞的增殖,为进一步深入研究HGF和NK4的功能、相互作用及作用机制奠定了基础,也为乳腺癌的靶向基因治疗提供了一个新的靶点。
Objective To construct recombinant adenovirus Ad5-hNK4-EGFP carrying enhanced green fluorescent protein (EGFP) tag and hNK4 gene and test its effect on the proliferation of breast cancer MDA-MB-231 cells. Methods The pcDNA3 / hNK4 plasmid was used as a template to amplify hNK4 gene with high-fidelity DNA polymerase and cloned into the adenovirus shuttle vector pAdTrack-CMV to construct the recombinant shuttle plasmid pAdTrack-hNK4. The shuttle plasmid was linearized with PmeⅠ, The recombinant plasmid pAdEasy-1 was homologously recombined in E. coli BJ5183 to obtain the recombinant adenovirus plasmid pAd5-hNK4-EGFP, which was linearized with PacⅠ and then transfected into AD-293 cells for packaging. The recombinant adenovirus Ad5-hNK4 The expression of NK4 gene in infected cells was detected by RT-PCR and Western blot. MTT assay was used to detect the expression of Ad5 Effect of hNK4-EGFP on proliferation of MDA-MB-231 cells induced by hepatocyte growth factor (HGF). Results Recombinant adenovirus plasmid pAd5-hNK4-EGFP was identified by restriction endonucleolytic enzyme digestion with restriction endonuclease PacⅠ. After packaging and 4 rounds of amplification, the recombinant adenovirus Ad5-hNK4-EGFP titer reached 1.5 × 1011 PFU / ml. PCR and Western blot showed that the recombinant adenovirus carrying NK4 gene can express in AD-293 cells; HGF can promote the proliferation of MDA-MB-231 cells; Ad5-hNK4-EGFP can antagonize the effect of HGF-induced MDA- Cell Proliferation. Conclusion The recombinant adenovirus Ad5-hNK4-EGFP expressing hNK4 gene was successfully constructed, which can antagonize HGF-induced proliferation of MDA-MB-231 cells, which lays the foundation for further study on the function, interaction and mechanism of HGF and NK4. , Also provides a new target for targeted gene therapy of breast cancer.