大鼠靶向α7nAchR基因shRNA真核表达载体及重组腺病毒构建

发布时间：2018-08-13 17:09

【摘要】：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。
[Abstract]:Objective to construct a rat nicotinic acetylcholine receptor 伪 7 subtype (伪 7nAchR) gene short hairpin RNA (shRNA) eukaryotic expression vector. Methods A DNA template primer AY318- AY857- AY444559AY318 857 encoding 伪 7nAchR gene shRNA was designed and synthesized, and ligated with the linearized plasmid vector pGenesil-1.1 pGenesil-1.2pGenesil-1.2pGenesil-1.3 to construct a recombinant plasmid vector encoding a 伪 7nAchR gene shRNA and two 伪 7nAchR gene shRNA. The recombinant plasmid vector was infected with the competent DH5a cells, the bacterial plasmid was extracted for specific enzyme digestion, and 1% agarose gel electrophoresis was used to identify whether the recombinant plasmid was successfully constructed. The AY318AY444559AY857shRNA expression frame was transferred from the recombinant plasmid pGenesil to the adenovirus pGSadeno plasmid expression vector by LR homologous recombination in vitro, and the recombinant adenovirus plasmid was constructed, and the DH5a cells were infected with the same method. The recombinant adenovirus pGSadeno plasmid was successfully constructed, the linearized recombinant adenovirus DNA was extracted and transfected into the packaging cell line HEK293, and the recombinant adenovirus supernatant with a titer of 1.0 脳 1010pfu/mL was obtained by amplification, and the recombinant adenovirus was infected with rat GH3 pituitary adenoma cells. The interference effect of 伪 7nAchR gene on recombinant adenovirus vector was evaluated by PCR reaction. Results the AY318AY857AY857AY444559AY318 857shRNA was successfully recombined into the plasmid vector, the recombinant adenovirus pGSadeno-shRNA was packaged successfully, the recombinant adenovirus infected HEK293 cells, and the green fluorescent protein was expressed under fluorescence microscope. After the recombinant adenovirus infected GH3 cells, the expression of 伪 7nAchR mRNA was significantly decreased, and pGSadeno-AY857shRNA interference was the most obvious (85%). Conclusion the eukaryotic expression vector of shRNA with infectious and interfering expression of rat 伪 7nAchR gene is successfully constructed, and pGSadeno-AY857shRNA has the strongest effect of gene interference.
【作者单位】：华中科技大学同济医学院附属同济医院中西医结合科;华中科技大学同济医学院附属同济医院中西医结合研究所;
【基金】：国家自然科学基金资助项目(No.81373872,No.81573785)
【分类号】：R3416

【相似文献】