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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。
Objective To construct the pSUPER-siCbp recombinant vector targeting Cbp gene by RNA interference technology and establish its efficient transfection expression system. Methods Oligonucleotide sequences targeting the Cbp gene were designed, cloned into a linearized pSUPER vector and identified. Jurkat cells were transfected by electrotransfection method. The expression of GFP gene was observed by fluorescence microscopy. The expression of Cbp in transfected cells was detected by RT-PCR and Western blot. The proliferation of Jurkat cells in each group before and after transfected with the recombinant vector was detected by MTT colorimetric assay. The level of interleukin 2 (IL-2) in the cell supernatant was measured by ELISA. Results Restriction endonuclease digestion analysis, DNA PCR and DNA sequencing confirmed that the recombinant plasmid pSUPER-siCbp correct sequence. The Jurkat cells transfected with recombinant plasmids could express green fluorescent protein. The expression of Cbp gene in Jurkat cells transfected with J2-pSUPER-siCbp recombinant plasmids was significantly lower than that in other groups. The cell proliferation and IL-2 secretion were higher than those in Jurkat cells each group. Conclusion The pSUPER-siCbp vector targeting human Cbp gene was constructed successfully. The expression of Cbp in transfected Jurkat cells was reduced, which laid the foundation for the future study on the interaction between CD59 and Cbp in T cell signal transduction pathway.