小鼠CDC26基因短发夹RNA重组腺病毒载体的构建与表达分析

Construction of Adenovirus Vector and Express Analysis of shRNA Against Mouse CDC26 Gene

目的:构建靶向CDC26基因的短发夹RNA(shRNA)重组腺病毒载体,干扰小鼠胚胎成纤维细胞(MEF)中CDC26的表达。方法:设计小鼠靶向CDC26基因的shRNA,插入穿梭质粒pENTRY-EF1a-EGFP-Mir30,通过Gateway系统获得重组腺病毒载体pAd-CDC26-shRNA;线性化后转染HEK293A细胞,进行病毒包装;用包装的重组腺病毒感染MEF,24 h后收集细胞,提取总RNA,逆转录后用荧光定量PCR法检测MEF内CDC26 mRNA的变化,以检测所设计shRNA的敲低效率。结果:构建的重组腺病毒载体pAd-CDC26-shRNA经酶切鉴定后正确;转染HEK293A细胞后8 d,观察到细胞病变及GFP的大量表达,表明重组腺病毒包装成功;感染MEF后,实时荧光定量PCR检测表明,细胞内CDC26 mRNA的水平较对照组敲低了73.14%,腺病毒注射7 d后,注射CDC26 shRNA的小鼠血糖较对照组明显降低。结论:构建了具有较高敲低效果的小鼠CDC26基因shRNA重组腺病毒,并且具有体内活性。

Objective： To construct the recombinant adenovirus vector of short hairpin RNA （shRNA） against CDC26 and explore the knocking-down expression of CDC26 in mouse embryonic fibroblast（MEF）. Methods： The short hairpin oligonucleotide and complementary strands targeting the consensus sequences of CDC26 was designed and inserted into the shuttle plasmid pENTRY-EFla-EGFP-Mir30, and then adenovirus vector pAd-CDC26-shRNA was constructed via Gateway system. The recombinant adenovirus vector was linearized and packaged by transfected into HEK293A cells with PEI, and then the virus particles were used to infect MEF cells. The total RNA was extracted from the 24 h cultured cells, and the relative expression of CDC26 mRNA was detected by subsequent reverse transcription and qPCR. Results： The shuttle vector pENTRY-CDC26-shRNA and adenovirus vector pAd-CDC26-shRNA were constructed successfully. Cytopathic effect and fluorescence detection indicated that the adenovirus was successfully packaged 8 d ＇following pAd-CDC26-shRNA infected MEF cells. The qPCR results indicated that the expression of CDC26 mRNA decreased 73.14%, and we also detected the blood level at the 7 d after injection, the results showed that the blood level of the mice injected with the shRNA adenovirus was apparently lowed than controls. Conclusion： We successfully constructed the recombinant CDC26 shRNA expressing adenovirus, and could obviously knockdown the expression of CDC26 mRNA and the mouse blood level was apparently lowered after the injection of adenovirus.