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为构建小鼠CD86启动子,并对其特异性调控能力进行鉴定。首先自C57BL/6J小鼠基因组中采用高保真PCR扩增小鼠CD86基因的启动子,经测序鉴定后,插入pCDNA3.1-EGFP重组质粒。然后分别转染小鼠来源细胞株:RAW264.7、NIH/3T3、P815、SP2/0、EL4细胞,观察各细胞内绿色荧光蛋白的表达。结果显示,小鼠CD86基因启动子的序列与GenBank报道一致;双酶切鉴定证实小鼠CD86启动子序列已经克隆入重组载体;小鼠CD86启动子能调控EGFP在巨噬细胞系RAW264.7细胞中表达,不能调控EGFP在NIH/3T3、P815、SP2/0和EL4非抗原提呈细胞内表达。说明成功构建小鼠CD86基因启动子,且该启动子具有特异性调控目的基因表达的活性。
To construct the mouse CD86 promoter and to identify its specific regulatory capacity. Firstly, the promoter of mouse CD86 gene was amplified by high-fidelity PCR from the genome of C57BL / 6J mice. After sequencing, the recombinant plasmid pCDNA3.1-EGFP was inserted into the plasmid. Then the mouse-derived cell lines were transfected with RAW264.7, NIH / 3T3, P815, SP2 / 0 and EL4 cells respectively to observe the expression of green fluorescent protein in each cell. The results showed that the sequence of mouse CD86 gene promoter was consistent with that reported in GenBank. Double enzyme digestion confirmed that the mouse CD86 promoter sequence had been cloned into the recombinant vector. The mouse CD86 promoter could regulate the expression of EGFP in macrophage cell line RAW264.7 EGFP can not regulate the expression of EGFP in NIH / 3T3, P815, SP2 / 0 and EL4 non-antigen presenting cells. The successful construction of mouse CD86 gene promoter, and the promoter has a specific regulation of the activity of the gene of interest.