摘要
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 a eukaryotic expression plasmid for mouse serine / arginine-rich protein specific kinase 2（SRPK2）,establish a He La cells for stable overexpression of SRPK2 and investigate the effect of SRPK2 overexpression on proliferation and apoptosis of He La cells.Methods SRPK2 c DNA fragment was amplified from mouse testis by reverse transcription polymerase chain reaction（RT-PCR）and inserted into plasmid p3 × Flag-CMV-14.The recombinant plasmid p3 × Flag-CMV-14-SRPK2 was transfected to He La cells,and the stably transfected cell lines were screened with G418,in which transcription of SRPK2 gene was determined by RT-PCR,while the expression of SRPK2 by Western blot.MTT and flow cytometry were used to detect the effects of SRPK2 on proliferation and apoptosis of He La cells respectively.Results Colony PCR,restriction analysis and sequencing proved that recombinant eukaryotic expression plasmid p3 × Flag-CMV-14-SRPK2 was constructed correctly.RT-PCR and Western blot proved that SRPK2 was stably overexpressed in He La cells.The proliferation activity of He La cells stably transfected with p3 × Flag-CMV-14-SRPK2 was significantly higher,while the apoptosis rate was significantly lower,than those transfected with p3 × FlagCMV-14 and those untransfected（P〈.05）.Conclusion The recombinant eukaryotic expression plasmid for SRPK2 was successfully constructed,and the He La cell lines for stable overexpression of SRPK2 was established.The overexpression of SRPK2 enhanced the proliferation and inhibited the apoptosis of He La cells.
出处
《中国生物制品学杂志》
CAS
CSCD
2016年第5期463-468,共6页
Chinese Journal of Biologicals
基金
山西省回国留学人员科研资助项目(2011-043)
