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目的构建pIRES-CD59融合基因真核表达载体,探讨重组CD59在Jurkat细胞增殖中的作用。方法 RT-PCR法选择性扩增编码CD59的基因片段,克隆入pIRES真核表达质粒。脂质体法将重组载体转染Jurkat细胞,通过G418筛选获得稳定表达CD59的细胞克隆,利用RT-PCR和Western blot检测细胞中CD59的表达,通过MTT法检测Jurkat细胞的增殖。结果经酶切和测序鉴定表明携带CD59基因的重组质粒构建成功,RT-PCR和Western blot结果显示转染Jurkat细胞的CD59基因高表达,MTT实验显示转染CD59重组质粒的Jurkat细胞增殖速度明显快于对照组(P<0.05)。结论 pIRES-CD59真核表达载体在转染细胞Jurkat中可高表达CD59分子,并可影响Jurkat细胞的增殖,为研究CD59的生物学作用奠定了基础。
Objective To construct eukaryotic expression vector pIRES-CD59 and explore the role of recombinant CD59 in proliferation of Jurkat cells. Methods The gene fragment encoding CD59 was amplified by RT-PCR and cloned into pIRES eukaryotic expression plasmid. The recombinant vector was transfected into Jurkat cells by lipofectamine. The cells stably expressing CD59 were screened by G418. The expression of CD59 was detected by RT-PCR and Western blot. The proliferation of Jurkat cells was detected by MTT assay. Results The recombinant plasmids carrying CD59 gene were successfully constructed. The results of RT-PCR and Western blot showed that CD59 gene was highly expressed in Jurkat cells. The MTT assay showed that the proliferation of Jurkat cells transfected with CD59 recombinant plasmid was significantly faster In the control group (P <0.05). CONCLUSION: The eukaryotic expression vector pIRES-CD59 can express CD59 molecules in Jurkat cells in vitro and can affect the proliferation of Jurkat cells, which lays the foundation for the study on the biological role of CD59.