大鼠靶向α7nAchR基因shRNA真核表达载体及重组腺病毒构建

Construction of Eukaryotic Expression Vector for Short Hairpin RNA Targeting Gene of Rat α7nAchR and Production of Recombinant Adenovirus

目的构建大鼠烟碱样乙酰胆碱受体α7亚型(α7nAchR)基因短发夹RNA(shRNA)真核表达载体。方法设计并合成编码α7nAchR基因shRNA的DNA模板引物AY318、AY857、AY444+559、AY318+857,并分别与线性化的质粒载体pGenesil-1.1、pGenesil-1.4、pGenesil-1.2,pGenesil-1.3连接,以构建可以编码1条α7nAchR基因shRNA和编码2条α7nAchR基因shRNA的重组质粒载体;重组质粒载体感染感受态的DH5a细胞,提取细菌质粒进行特定酶切,并用1%的琼脂糖凝胶电泳以鉴定重组质粒是否构建成功;用LR体外同源重组法将AY318、AY444+559、AY857shRNA表达框分别从重组质粒pGenesil转移至腺病毒pGSadeno质粒表达载体上,构建重组腺病毒质粒;用相同的方法感染DH5a细胞,并鉴定重组腺病毒pGSadeno质粒是否构建成功;提取线性化的重组腺病毒DNA并转染包装细胞HEK293,经放大培养获得滴度为1.0×1010pfu/mL的重组腺病毒上清;将重组腺病毒感染大鼠GH3垂体瘤细胞,以PCR反应鉴定重组腺病毒载体的α7nAchR基因干扰效果。结果通过酶切片段及其大小,初步判断AY318、AY857、AY444+559、AY318+857shRNA成功重组入质粒载体,重组腺病毒pGSadeno-shRNA包装成功;重组腺病毒感染HEK293细胞,在荧光显微镜下有绿色荧光蛋白表达;重组腺病毒感染GH3细胞后α7nAchR mRNA表达显著减少,且pGSadeno-AY857shRNA干扰效果最明显(85%)。结论具有感染力和干扰大鼠α7nAchR基因表达的shRNA真核表达载体构建成功,且pGSadeno-AY857shRNA具有最强的基因干扰效果。

Objective To construct eukaryotic expression vector for shRNA targeting gene of ratα7nAchR.Methods We designed and synthesized DNA templates（AY318,AY857,AY444＋559,AY318＋857）which would encode shRNA ofα7nAchR.To construct recombinant plasmid vector which would encode one or two shRNA ofα7nAchR,DNA templates listed were connected with linearized plasmid vector（pGenesil-1.1,pGenesil-1.4,pGenesil-1.2,pGenesil-1.3）.To identify the recombinant plasmid vectors,DH5 acells were infected with the recombinant plasmid vectors,and plasmids from DH5 awere extracted and electrophoresed in 1% agarose gel.To construct recombinant adenovirus plasmid,LR homologous recombination method in vitro was used to transfer AY318,AY444＋559,AY857 shRNA expression cassettes of shRNA from pGenesil to pGSadeno from adenovirus plasmids,and a similar method was used to identify the recombinant adenovirus plasmid of pGSadeno.To obtain specific titer of recombinant adenovirus,we extracted DNA from the recombinant adenovirus and infected HEK293 cells increasingly.To identify the effect ofα7nAchR gene silencing,we infected GH3 cells with the recombinant adenovirus and PCR was applied to detect the expression ofα7nAchR mRNA in different groups（pGSadeno＋AY318shRNA,pGSadeno ＋AY857shRNA,pGSadeno＋AY444＋559shRNA,pGSadeno＋HK shRNA and Normal group）.Results We succssefully constructed the recombinant plasmids（pGenesil-AY318,AY857,AY318＋857and AY444＋559shRNA）and pGSadeno shRNA.HEK293 cells expressed green fluorescent protein under infection of recombinant adenovirus.The mRNA expression ofα7nAchR was decreased in GH3 cells infected with the recombinant adenovirus（pGSadenoAY318,AY857,AY444＋559shRNA）,and pGSadeno-AY857 shRNA showed the best effect ofα7nAchR gene silencing.Conclusion We have constructed the recombinant adenovirus of pGSadeno-α7nAchR-shRNA successfully,and pGSadeno-AY857 shRNA shows the best effect of gene interference.