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目的构建人类CD40膜蛋白siRNA真核表达质粒,观察其能否抑制CA46细胞CD40表达。方法合成2条编码发夹siRNA序列的单链DNA,克隆到pSilenCircle载体中,构建成含目的基因片段的重组质粒———siCD40/pSilenCircle,在polⅢU6基因启动子控制下表达siRNA。同样方法构建相对应的编码反义RNA以及无关基因的重组质粒antiCD40/pSilenCircle和siFly/pSilenCircle,以作比较和对照。PCR制备CD40-SECs(CD40-siRNA表达框架)。以脂质体FuGene 6为介质瞬时转染CA46细胞,利用流式细胞技术检测细胞膜CD40的表达。结果(1)成功构建了CD40发夹siRNA真核表达质粒siCD40/pSilenCircle、CD40反义RNA真核表达质粒antiCD40/pSilenCircle和无关基因重组质粒siFly/pSilenCircle;(2)PCR成功制备CD40-SEC;(3)与siFly/pSilenCircle转染组相比较,siCD40/pSilenCircle、antiCD40/pSilenCircle和CD40-SEC转染组的CA46细胞CD40的表达均明显减少。结论CD40发夹siRNA真核表达质粒siCD40/pSilenCircle和CD40-SEC可有效地抑制CA46细胞CD40分子的表达。RNA干扰技术可望作为一种有效的调控基因功能的工具。
Objective To construct the eukaryotic expression plasmid of human CD40 membrane protein and observe whether it can inhibit the expression of CD40 on CA46 cells. Methods Two single-stranded DNA encoding the hairpin siRNA sequence were synthesized and cloned into pSilenCircle vector to construct a recombinant plasmid containing the target gene fragment, siCD40 / pSilenCircle. SiRNA was expressed under the control of pol Ⅲ U6 promoter. The same method was used to construct the corresponding recombinant anti-CD40 / pSilenCircle and siFly / pSilenCircle encoding antisense RNA and unrelated genes for comparison and control. PCR to prepare CD40-SECs (CD40-siRNA expression framework). The transient transfection of CA46 cells with lipofectamine FuGene 6 was used to detect the expression of CD40 by flow cytometry. Results (1) CD40 hairpin RNAi eukaryotic expression plasmid siCD40 / pSilenCircle, CD40 antisense RNA antiCD40 / pSilenCircle and unrelated gene siFly / pSilenCircle were constructed successfully. (2) CD40-SEC was successfully prepared by PCR. 3) Compared with siFly / pSilenCircle transfection group, the expression of CD40 in CA46 cells of siCD40 / pSilenCircle, antiCD40 / pSilenCircle and CD40-SEC transfection group were significantly reduced. Conclusion CD40 hairpin siRNA eukaryotic expression plasmid siCD40 / pSilenCircle and CD40-SEC can effectively inhibit the expression of CD40 in CA46 cells. RNA interference technology is expected as an effective tool to regulate gene function.