pIRES2-EGFP-BCL11B真核表达载体体外表达的动态分析

Dynamic analysis of in vitro expression of B-cell lymphoma/leukemia 11B gene by eukaryotic expressive plasmid pIRES2-EGFP-BCL11B

目的:研究真核表达载体pIRES2-EGFP-BCL11B(B细胞白血病/淋巴瘤11B基因)在K293(人胚肾细胞株)和Raji(人Burkitt淋巴瘤细胞株)细胞中的表达情况。方法:分别利用脂质体和核转染技术将真核表达载体pIRES2-EGFP-BCL11B(pBCL11B)转染至K293和Raji细胞中,并设立空质粒组(pI2)和空转染组(MOCK)作为对照。通过荧光显微镜和流式细胞仪检测转染后48 h各组EGFP的表达,确定转染效率。通过荧光实时定量PCR(Taqman)技术检测转染后24 h各组BCL11B mRNA的表达情况,通过Western blotting技术检测转染后48 h各组BCL11B蛋白的表达情况。高分辨率活细胞成像系统动态观察转染后36-72 h重组质粒在Raji细胞增殖过程中的表达。结果:K293细胞pBCL11B、pI2和MOCK组的转染效率分别为(71.23±4.62)%、(70.45±3.58)%和(0.30±0.10)%,而Raji细胞各组的转染效率分别为(23.12±5.94)%、(22.48±6.25)%和(0.30±0.10)%。实时定量PCR和Western blotting结果显示转染后pBCL11B组在mRNA和蛋白水平均能检测到BCL11B基因的有效表达,BCL11B的表达量均明显高于pI2组和MOCK组(P<0.05)。活细胞追踪显示转染后36-72 h重组质粒在细胞中可稳定表达且转染后的细胞可以正常分裂增殖。结论:系统分析了真核表达载体pIRES2-EGFP-BCL11B转染至K293和Raji细胞的表达变化特点,转染后可以检测到该基因的有效上调,研究结果为下一步转染正常人T细胞奠定了基础。

AIM： To analyze the ability of eukaryotic expressive plasmid pIRES2-EGFP-BCL11B to express B-cell lymphoma/leukemia 11B（BCL11B） gene in K293 and Raji cells.METHODS： The eukaryotic expressive plasmid pIRES2-BCL11B-EGFP（pBCL11B） was transfected into K293 cells and Raji cells by the techniques of Lipofectamine and Nucleofector transfection,respectively.Empty plasmid（pI2） and mock-transfection（MOCK） were used as controls.The transfection efficiency was examined by the methods of fluorescence microscopy and flow cytometry 48 h after transfection.The mRNA expression of BCL11B was analyzed at 24 h by real-time quantitative PCR with Taqman technique.The protein levels of BCL11B were determined at 48 h by Western blotting.The expression of recombinant plasmid during proliferation of Raji cells transfected with pIRES2-EGFP-BCL11B were observed by the high-resolution live cell imaging system from 36 h to 72 h.RESULTS： The transfection efficiency in pBCL11B,pI2 and MOCK groups in K293 cells was（71.23±4.62）%,（70.45±3.58）% and（0.30±0.10）%,respectively.Simultaneously,that in Raji cells was（23.12±5.94）%,（22.48±6.25）% and（0.30±0.10）%,respectively.Compared with pI2 and MOCK controls,the mRNA and the corresponding proteins of the BCL11B gene were effectively in up-regulated pBCL11B group（P0.05）.The results of high-resolution live cell imaging system showed that the recombinant plasmid stably expressed EGFP protein from 36 h to 72 h after transfection,and the transfected cells proliferated normally.CONCLUSION： BCL11B gene is effectively up-regulated in K293 and Raji cells after transfected with pIRES2-BCL11B-EGFP eukaryotic expressive plasmid,indicating a useful way for subsequent step in human T-cells transfection.