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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 an eukaryotic expression vector of short hairpin RNA (shRNA) of α7nAchR gene of rat nicotinic acetylcholine receptor. Methods DNA template primers AY318, AY857, AY444 + 559 and AY318 + 857 were designed and synthesized. The linearized plasmid vectors pGenesil-1.1, pGenesil-1.4, pGenesil-1.2 and pGenesil-1.3 were designed and synthesized. Construction of a recombinant plasmid vector that can encode a shRNA of a7nAchR gene and two shRNAs encoding a7nAchR gene. The recombinant plasmid vector was infected into competent DH5a cells, and bacterial plasmids were extracted for specific digestion and electrophoresed with 1% agarose gel to identify recombinant The recombinant plasmid pGenesil was used to transfer the AY318, AY444 + 559 and AY857shRNA expression plasmids into the adenovirus pGSadeno plasmid expression vector by LR in vitro homologous recombination to construct the recombinant adenovirus plasmid. The same method was used to infect DH5a cells , And to confirm whether the recombinant adenovirus pGSadeno plasmid was successfully constructed. The linearized recombinant adenovirus DNA was extracted and transfected into the packaging cell HEK293, and the recombinant adenovirus supernatant with a titer of 1.0 × 1010pfu / mL was obtained by amplification culture. The recombinant adenovirus Infection of rat GH3 pituitary tumor cells by PCR reaction identified recombinant adenovirus vector α7nAchR gene interference effect. Results The recombinant plasmid AY318, AY857, AY444 + 559 and AY318 + 857 were successfully cloned into plasmid vector and the recombinant adenovirus pGSadeno-shRNA was successfully packaged. The recombinant adenovirus was infected into HEK293 cells under a fluorescence microscope The expression of α7nAchR mRNA was significantly decreased in GH3 cells infected with recombinant adenovirus, and the effect of pGSadeno-AY857 shRNA was the most obvious (85%). CONCLUSION: The eukaryotic expression vector of shRNA with the ability of infecting and interfering with α7nAchR gene expression in rats has been successfully constructed, and pGSadeno-AY857shRNA has the strongest gene interference effect.