CD40发夹siRNA真核表达质粒与siRNA表达框架对CA46细胞CD40表达的影响  

Inhibition of CD40 Expression on CA46 Cell Lines by Hairpin siRNA Eecombinant Plasmid and siRNA Expression Cassette

作　　者：陈凌[1] 郑祥雄[1] 

机构地区：[1]福建医科大学附属协和医院临床免疫研究所,福州350001

出　　处：《福建医科大学学报》2006年第4期323-326,共4页Journal of Fujian Medical University

基　　金：福建省自然科学基金资助项目(C0310019)

摘　　要：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 human CD40 hairpin siRNA recombinant plasmid and prepare siRNA expression cassette, investigate their effects on CD40 expression. Methods Two CD40 sequence-specific DNA oligonucleotides were synthesized. Then the oligos were cloned in the plasmid to form siCD40/pSilenCircle recombinant plasmid that expresses hairpin siRNAs under the control of the pol Ⅲ U6 gene promoter. At the same time, the same method was used to construct the antiCD40/pSilenCircle encoding the antisense CD40 RNA and siFly/pSilenCircle encoding siRNA against luciferase. The recombinant plasmids were identified by restriction enzyme digestion and sequencing. Prepare siRNA expression cassettes by PCR. The plasmids and siRNA expression cassettes were transfected into CA46 cell line using FuGene6. Results （1）siCD40/pSilenCircle, antiCD40/pSilenCircle and siFly/pSilenCircle had been constructed successfully. （2）CD40 siRNA expression cassettes had been prepared. （3）CD40 expressions on siCD40/pSilenCircle-, antiCD40/pSilenCircle-, and siRNA expression cassettes-transfected CA46 cells were significantly reduced, as compared with those on siFly/pSilenCircle-transfected CA46 cells （P〈0.01, P〈0.05 and P〈0.01, respectively）. Conclusion Constructed both siCD40/pSilenCircle and antiCD40/pSilenCircle and prepared CD40 siRNA expression cassettes can significantly inhibit CD40 expression. RNA interfering technology may serve as an effective means of gene regulation.

关 键 词：RNA interference RNA small interfering 抗原 CD40 质粒 CA46细胞 

分 类 号：R392[医药卫生—免疫学]