大鼠缝隙连接蛋白Connexin 43的shRNA重组腺病毒载体的构建和鉴定

目的:构建缝隙连接蛋白connexin 43(CX43)特异性shRNA重组腺病毒载体,为应用基因沉默技术从转录后水平进行基因治疗研究奠定基础。方法:合成CX43发夹样shRNA的DNA单链一对和对照单链scramble一对,退火后将其克隆至带有增强型绿色荧光蛋白(eGFP)报告基因的穿梭质粒pAd shRNA/H1,得到pAd shRNA/H1-CX43和pAd shRNA/H1-scramble。分别酶切及DNA测序鉴定后,经Pme I线性化后转化入BJ5187-AD-1感受态细菌,在细菌内进行同源重组构建含目的基因的重组腺病毒质粒载体。用Pac I酶切鉴定重组菌。将Pac I酶切重组的两种腺病毒质粒载体后,经脂质体转化293细胞进行包装得到重组的腺病毒颗粒,并通过293细胞绿色荧光表达反应重组腺病毒表达情况。将重组的腺病毒Ad-Cx43 shRNA和对照病毒Ad-scramble shRNA分别感染大鼠原代培养的星形胶质细胞,并检测星形胶质细胞CX43的表达。结果:证实穿梭质粒的以及重组腺病毒质粒均构建正确,腺病毒质粒转染293细胞后48 h即可见绿色荧光蛋白表达,10 d后全部细胞表达绿色荧光,并有半数细胞飘起,收获病毒感染培养星形胶质细胞后48 h,均有绿色荧光表达,经感染Ad-Cx43 shRNA的星形胶质细胞CX43的表达明显低于感染对照病毒Ad-scramble shRNA的星形胶质细胞。结论:成功构建表达大鼠CX43特异性发夹状shRNA的重组腺病毒载体,将为应用基因沉默技术研究CX43在神经系统疾病尤其是缺血性脑损伤中的作用提供依据。

Construction and Identification of Recombinant Adenovirus Expressing the shRNA of Rat Connexin-43

Objective：To construct the recombinant adenovirus vector with rat gene connexin 43（shRNA,short hairpin RNA） by pAdEasy-1 vector.Methods： CX43 shRNA was obtained after a couple of single-stranded DNA of CX43 shRNA were synthesized and annealed.Then the CX43 shRNA was cloned to a shuttle plasmid pAd shRNA/H1.After the product was identified pAd shRNA/H1-CX43 was linearized by PmeI to mediate homologous recombination with pAdEasy-1 in pAdEasy-1 host bacteria BJ5187-AD-1.The positive clone was identified by enzyme digestion and PCR analysis.After linearized by PacI,the recombinant adenovirus DNA Ad-Cx43 shRNA was transfected into 293 cells for packaging and amplification.Western blotting was used to detect the expression of CX43 protein in cultured astrocytes infected with the adenovirus.Results： The pAd shRNA/H1-CX43 and Ad-Cx43 shRNA plasmids had been successfully constructed as verified by PCR analysis,enzyme digestion and DNA sequence analysis.PCR analysis confirmed successful packaging of the recombinant adenovirus Ad-Cx43 shRNA in 293 cells.CX43 protein expression was decreased significantly in the cultured astrocytes after infection by the recombinant virus.Conclusion： We have successfully constructed the recombinant adenovirus Ad-Cx43 shRNA targeting CX 43 gene,which provides a basis for investigating the role of CX43 in neuroprotection after cerebral ischemic injury using RNA interference.