新锌指蛋白基因Zfp637真核表达质粒的构建及对乳腺癌EMT6细胞的作用

Construction of Eukaryotic Expression Plasmid for a Novel Zinc Finger Protein Gene Zfp637 and Its Effect on Breast Carcinoma Cell EMT6

目的构建锌指蛋白Zfp637真核表达质粒及稳定转染过表达细胞,并观察Zfp637基因对肿瘤细胞生长增殖的影响。方法以正常小鼠脾脏组织cDNA作为模板扩增Zfp637DNA,定向插入到pEGFP-C3真核表达质粒中,酶切片段分析和测序鉴定以确定成功构建pEGFP-Zfp637重组质粒,通过脂质体介导pEGFP-Zfp637重组质粒转染小鼠乳腺癌株EMT6细胞,并且通过G418筛选出稳定转染细胞(Zfp637-EMT6)。荧光显微镜下对Zfp637进行亚细胞定位;RT-PCR检测Zfp637-EMT6细胞中Zfp637基因表达;同时观察细胞生长状况,MTT法测定Zfp637-EMT6细胞的增殖活性。结果重组质粒经鉴定证实具有Zfp637全长cDNA序列,所建立的Zfp637-EMT6细胞能稳定过表达Zfp637基因,并且其细胞生长增殖显著增加。结论成功构建pEGFP-Zfp637真核表达质粒,显示了Zfp637能促进肿瘤细胞增殖,为进一步研究Zfp637基因的功能奠定了基础,并将为恶性肿瘤的基因治疗提供新靶点。

Objective To construct the eukaryotic expression plasmid and establish stably transfected cell line, which contained the code gene of zinc finger protein637 （Zfp637）, and to observe the effect of Zfp637 gene to the proliferation of tumor cells. Methods The Zfp637 DNA was amplified from the template of normal spleen tissue cDNA and cloned into the eukaryotic expression vector pEGFP-C3. The recombinant plasmid, named as pEGFP-Zfp637, was determined by restriction enzyme and sequencing analyses. Next the pEGFP-Zfp637 recombinant plasmid was transferred into mouse breast carcinoma EMT6 cells with lipofectamine 2000, and the stably transfected cells were selected by G418 （named Zfp637-EMT6）. The growth condition of cells was observed,and subcellular localization of Zfp637 gene was located by fluorescence microscope at the same time. The Zfp637 mRNA expression in the transfected cells was detected by RT-PCR, and the proliferation of such cells was measured by MTT. Results The analysis confirmed that the recombinant pEGFP-Zfp637 contained the Zfp637 full-length cDNA. The Zfp637 mRNA was over-expressed stably in Zfp637-EMT6. The growth of Zfp637- EMT6 was increased obviously when compared with the negative control group and blank group. Conclusion The recombinant pEGFP-Zfp637 has been constructed successfully,and the expression of the Zfp637 gene can promote the proliferation of cells. This recombinant eukaryotic expression plasmid can be used in advanced studies in the biological effects of Zfp637 and anti-tumor gene therapy.