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目的构建小鼠丝氨酸/精氨酸蛋白特异激酶2(serine/arginine-rich protein specific kinase 2,SRPK2)基因真核表达质粒,建立稳定过表达SRPK2的He La细胞系,并探讨SRPK2过表达对He La细胞增殖和凋亡的影响。方法利用RT-PCR从小鼠睾丸组织中扩增SRPK2基因,与p3×Flag-CMV-14质粒连接,构建重组真核表达质粒p3×Flag-CMV-14-SRPK2,转染He La细胞,通过G418筛选获得稳定转染细胞系。采用RT-PCR检测稳定转染He La细胞系中SRPK2基因的转录,Western blot检测SRPK2的表达;MTT法和流式细胞术分别检测SRPK2过表达对He La细胞增殖和凋亡的影响。结果菌落PCR、双酶切及DNA测序表明重组真核表达质粒p3×Flag-CMV-14-SRPK2构建正确;RT-PCR和Western blot分析表明SRPK2基因在He La细胞中稳定过表达;p3×Flag-CMV-14-SRPK2稳定转染组细胞增殖活力明显高于p3×Flag-CMV-14转染组和未转染组(P<0.05),细胞凋亡率明显低于上述两组(P<0.05)。结论成功构建了SRPK2基因的真核表达质粒,建立了稳定过表达SRPK2的He La细胞系,SRPK2过表达可增强He La细胞的增殖活力,而抑制其凋亡。
Objective To construct the eukaryotic expression plasmid of mouse serine / arginine-rich protein specific kinase 2 (SRPK2) gene and establish a He La cell line stably overexpressing SRPK2, and to explore the effect of SRPK2 overexpression on He La cell proliferation and apoptosis. Methods The SRPK2 gene was amplified from mouse testes by RT-PCR and ligated into plasmid p3 × Flag-CMV-14 to construct recombinant eukaryotic expression vector p3 × Flag-CMV-14-SRPK2. The recombinant plasmid was transfected into HeLa cells and transfected by G418 Screening for stable transfected cell lines. The transcription of SRPK2 gene was detected by RT-PCR in stably transfected HeLa cells and the expression of SRPK2 was detected by Western blot. The effect of SRPK2 overexpression on proliferation and apoptosis of HeLa cells was detected by MTT assay and flow cytometry. Results The results of colony PCR, double enzyme digestion and DNA sequencing showed that the recombinant eukaryotic expression plasmid p3 × Flag-CMV-14-SRPK2 was constructed correctly. RT-PCR and Western blot showed that SRPK2 gene was overexpressed in HeLa cells. P3 × Flag The cell viability of CMV-14-SRPK2-stably transfected group was significantly higher than that of p3 × Flag-CMV-14 transfected group and untransfected group (P <0.05), and the apoptosis rate was significantly lower than that of the above two groups (P < 0.05). Conclusion The eukaryotic expression plasmid of SRPK2 gene was successfully constructed and the He La cell line stably overexpressed by SRPK2 was established. Overexpression of SRPK2 enhanced the viability of He La cells and inhibited their apoptosis.