摘要
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。
pIRES-CD59 fusion gene eukaryotic expression vector was constructed in order to discuss therole of CD59 gene in Jurkat cell proliferation.CD59 gene was amplified by RT-PCR and inserted into theeukaryotic expression plasmid-pIRES.The vector containing the object gene was transfected into Jurkat cells byliposome,while steady cells cloned could get through G418 screening.CD59 mRNA and protein levels weredetected by RT-PCR and Western blot;Jurkat cell proliferation was analyzed by MTT.The eukaryotic expressionvector containing CD59 gene was constructed correctly,which was confirmed by restriction endonuclease digestionand DNA sequencing analysis.RT-PCR and Western blot indicated that CD59 genes are highly expressed intransfected Jurkat cells,while MTT showed that the group of transfected restructuring plasmid could significantlyaffect Jurkat cell proliferation more than the control groups（P0.05）.These results showed that the pIRES-CD59eukaryotic expression vector can encode the expression of CD59 in Jurkat cells,and promote the cell proliferation,which provides a basis for the study of biological effects of CD59.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2011年第3期228-231,共4页
Immunological Journal
基金
国家自然科学基金资助项目(30972677)
