人ICOS胞外区腺病毒载体的构建与鉴定

Construction and identification of human ICOS extracellular domain gene recombinant adenovirus

目的利用细菌内同源重组法构建含ICOS胞外区基因的重组腺病毒。方法用基因工程的方法将ICOS胞外区的cDNA片段插入穿梭质粒pAdTrack-CMV中,形成转移质粒pAdtrack-cmv-ICOS,将之PmeⅠ酶切线性化后与腺病毒基因组质粒pAdEasy-1共转化大肠杆菌BJ5183,同源重组质粒PacⅠ酶切鉴定后用脂质体转染293细胞,包装成重组体腺病毒颗粒Ad-ICOS。采用PCR方法对重组腺病毒颗粒进行鉴定,利用穿梭质粒pAdTrack-CMV中带有GFP报告基因,对病毒滴度和感染效率进行监测。通过Western Blot检测重组腺病毒感染后的293细胞上清中的ICOS胞外区蛋白。结果获得了重组人ICOS胞外区腺病毒载体颗粒。PCR检测表明重组腺病毒颗粒含有目的基因,滴度为2.1×1010pfu/ml。Western Blot检测到阳性目的条带。结论细菌内同源重组法是一种高效、简便、快捷的重组体腺病毒载体制备方法。所制备的重组体腺病毒Ad-ICOS在体外能有效表达相应的基因产物,为今后对ICOS胞外区的深入研究奠定了基础。

Objective To construct the recombinant adenovirus of human ICOS extracellular domain gene by using the method of homologous recombination in bacteria.Methods Human ICOS extracellular domain cDNA fragment was obtained from tonsil by nest RT-PCR.Then human ICOS extracellular domain cDNA fragment was subcloned into the shuttle plasmid pAdTrack-CMV.The resulting plasmid pAdtrack-cmv-ICOS was linearized with PmeⅠ and cotransformed into BJ5183 cells with adenovirus genomic plasmid of pAdEasy-1.The DNA of identified recombinant plasmid was digested with PacⅠand transfected to 293 cells to package recombinant adenovirus particles.The resulting recombinant adenovirus was identified by PCR and expression of ICOS extracellular domain protein secreting to supernatant by western blot.The titre was measured with the aid of GFP expression.Results Obtained the recombinant adenoviral Ad-ICOS.PCR and western blot test indicated the recombinant adenovirus contained the insert of human ICOS extracellular domain cDNA and expressed the ICOS extracellular domain protein.The titre of purified recombinant adenovirus was 2.1×10^(10)pfu/ml.Conclusion The method of homologous recombination in bacteria is a convenient and efficient method to make recombinant adenovirus,the resulting Ad-ICOS can effectively madiate target gene expression in cultured cells and paves a sound foundation for further study.