人分化抑制因子3重组腺病毒的制备及鉴定

Preparation and identification of recombinant adenovirus carrying human inhibitors of differentiation 3

目的构建人细胞分化抑制因子3(Id3)重组腺病毒载体,制备并鉴定人Id3重组腺病毒,为研究Id3基因在细胞中的表达及其对细胞功能的影响奠定基础。方法以pUC57-Id3为模板扩增人Id3基因,克隆到质粒pUC18中,pUC18-Id3经EcoR I和HindⅢ双酶切补平之后与载体pAxCAwtit连接,构建重组粘粒载体pAxCAwtit-Id3,包装试剂盒包装重组粘粒,转染大肠埃希菌,鉴定正确后大量制备重组pAxCAwtit-Id3,经酶切线性化后,用脂质体法转染HEK293A细胞,包装成重组腺病毒Ad-Id3,制备高滴度的重组腺病毒Ad-Id3,利用TCID50法计算重组腺病毒滴度,经RT-PCR及western blot检测Id3基因的表达。结果Id3基因成功克隆到腺病毒载体中,重组腺病毒滴度为1.8×1010PFU/ml,用RT-PCR检测到Id3基因的转录产物,westernblot检测到Id3在A549细胞中的高水平表达。结论成功构建Ad-Id3重组腺病毒载体,并在A549细胞中得到高效表达。

Objective To construct recombinant adenoviral vector carrying human inhibitors of differentiation 3 （Id3）. Methods Id3 gene was amplified from recombinant plasmid pUC57-Id3 and cloned into the plasmid pUC18. The recombinant plasmid pUClS-Id3 was digested by EcoR I and Hind III and converted into blunt-end. The recombinant cosmid pAxCAwtit-Id3 was constructed by ligating double-enzyme-cut products and cosmid vector pAxCAwtit. Packaging recombinant cosmid was performed by using k-packaging kit, infecting E. coil and preparing the recombinant pAxCAwtit-Id3. HEK295A cells were transfected by the linearized recombinant pAxCAwtit-Id3. High titer recombinant adenovirus was prepared and determined for titers by TCIDs0. The expression of Id3 gene was identified western blotting. Results ld3 gene was successfully cloned to adenovirus vector, and the recombinant adenovirus reached a titer of 1.8 × 10^10 PFU/ml. Meanwhile, RT-PCR proved the transcription of ld3 gene, and western blotting proved the high expression of Id3 gene in A549 cells. Conclusion The recombinant adenovirus containing ld3 gene was constructed successfully and Id3 gene was highly expressed.