靶向大鼠PALM3基因的RNA重组腺病毒载体构建及鉴定  

Construction and identification of recombinant adenovirus expressing short hairpin RNA of rat paralemmin-3

作　　者：付玉梅[1] 陈旭昕[2] 韩志海[1,2] 

机构地区：[1]安徽医科大学海军临床学院,北京100037 [2]海军总医院呼吸科,北京100037

出　　处：《西部医学》2015年第8期1124-1127,共4页Medical Journal of West China

基　　金：国家自然科学基金(81300050);海军总医院创新培养基金(CXPY201417)

摘　　要：目的构建靶向大鼠paralemmin-3(PALM3)基因的短发夹shRNA(RNA)重组腺病毒载体,干扰大鼠Ⅱ型肺泡上皮细胞(AE2cells)中PALM3的表达。方法设计大鼠靶向PALM3基因的shRNA,插入穿梭质粒pGV120-EGFP,行PCR、测序鉴定;将穿梭质粒和骨架质粒共转293细胞,进行重组腺病毒Ad-palm3-shRNA-EGFP的包装,并采用TCID50法测定病毒感染滴度;用此病毒感染大鼠AE2细胞,Western blot法检测细胞内PALM3蛋白表达。结果穿梭质粒pGV120-palm3-shRNA-EGFP经PCR及测序鉴定证实构建成功,进一步构建了表达大鼠PALM3基因shRNA的重组腺病毒载体Ad-palm3-shRNA-EGFP,重组腺病毒感染滴度为3×1010 pfu/mL,感染大鼠AE2细胞后显著抑制目的蛋白表达(P<0.05)。结论本实验成功构建了针对大鼠PALM3基因的shRNA重组腺病毒,显著下调了大鼠AE2细胞中PALM3的表达,为进一步探索PALM3在炎性失控性疾病中的作用奠定基础。Objective To construct the recombinant adenovirus vector （Ad. V） of short hairpin RNA （shRNA） against rat paralemmin-3 （PALM3） gene and identify the effects of down-regulation of recombinant Ad. V in rat alveolar epithelial 2 cells （AE2 cells）. Methods The short hairpin oligonucleotide and complementary strand targeting the con- sensus sequences of PALM3 were designed and inserted into the shuttle plasmid pGV120-EGFP. The pGV120-palm3- shRNA-EGFP was identified by PCR and DNA sequencing. Recombinant viruses were generated by using 293 packaging cells co-transfeeted with the shuttle plasmid and the framing plasmid. The titer of Ad-palm3-shRNA-EGFP was deter- mined by means of 50% tissue culture infectious dose. And then the recombinant adenovirus Ad-palm3-shRNA-EGFP was used to infect the rat AE2 cells and the protein expression of PALM3 was detected by Western blot. Results The PCR and DNA sequencing results demonstrated that the shuttle plasmid pGV120-palm3-shRNA-EGFP was successfully constructed. Cytopathic effect and fluorescence detection indicated that the adenovirus was successfully packaged after co- transfection. The titer of Ad-palm3-shRNA-EGFP was 3×10^10 pfu/mL. The PALM3 protein expression in the rat AE2 cells was significantly decreased. Conclusion The recombinant adenovirus vectors target to rat PALM3 are successfully constructed and packaged and it could obviously knockdown the expression of PALM3 in the rat AE2 cells, which offers a basis for further research on the role of PALM3 in inflammatory diseases.

关 键 词：短发夹RNA paralemmin-3 重组腺病毒载体 RNA干扰 急性肺损伤 

分 类 号：R383.16[医药卫生—医学寄生虫学]