中文摘要：

目的采用RNA干扰技术构建针对Cbp基因的pSUPER-siCbp重组载体,以建立其高效转染表达体系。方法设计针对Cbp基因的寡核苷酸序列,克隆至线性化的pSUPER载体中并进行鉴定。采用电转染方法转染Jurkat细胞,荧光显微镜观察GFP基因表达,并行RT-PCR和Western blot检测转染细胞中Cbp的表达。利用噻唑蓝（MTT）比色法检测转染重组载体前后各组Jurkat细胞的增殖效应;应用ELISA检测细胞上清中白介素2（IL-2）的变化水平,比较各组差异。结果经过限制性内切酶双酶切分析、DNA PCR和DNA测序鉴定,证实重组质粒pSUPER-siCbp的序列正确。转染重组质粒的Jurkat细胞,可表达绿色荧光蛋白,转染J2-pSUPER-siCbp重组质粒的Jurkat细胞中Cbp基因表达量明显低于其他各组;其细胞增殖能力,IL-2分泌高于其他各组。结论成功构建了靶向人Cbp基因的pSUPER-siCbp载体,转染Jurkat细胞中Cbp表达降低,为以后的CD59与Cbp在T细胞信号转导通路中相互作用研究奠定了基础。

英文摘要：

This study aimed to construct recombinant vectors Cbp-pSUPER targeting Cbp gene by RNA interference technology and to gain a high transfection efficiency cell line.Two pairs of shRNA were designed,synthesized,and cloned into the linearized pSUPER vector.After identified by DNA PCR,restriction endonuclease digestion and DNA sequencing analysis,the recombinant vectors Cbp-pSUPER were transfected into Jurkat cell by the recombinant plasmids using electric transfection methods.The GFP expression was observed by the fluorescence microscopy,which confirmed that the recombinant vectors had transfected into Jurkat cells.RT-PCR and Western blot revealed that the level of Cbp in Jurkat cells transfected with the recombinant plasmid J2-pSUPER-siCbp were significantly lower than the other groups,while MTT colorimetry and ELISA illustrated that its cell proliferation and IL-2 level were increased compared with others（P 0.05）.The result indicated that the recombinant vectors are successfully constructed and the expression of Cbp is significantly reduced in Jurkat cell transferred with of J2-pSUPER-siCbp,which should lay a foundation for further study of the reciprocity of CD59 and Cbp in T cell signal transduction.