摘要
目的构建GM-CSF基因重组腺病毒穿梭载体并探讨XbaI甲基化位点在构建中的应用,为构建GM-CSF基因重组腺病毒及更方便地利用XbaⅠ多克隆位点打下基础。方法体外分离小鼠脾细胞,经ConA刺激,用含两种不同的XbaⅠ位点毗邻碱基的引物RT-PCR获取小鼠GM-CSF基因,经XbaⅠ及XbaⅠ双酶切后分别定向克隆入pAdTrack-CMV重组腺病毒穿梭载体中,比较两种重组载体酶切鉴定的效率并通过测序验证。结果成功逆转录得到小鼠GM-CSF基因并克隆入pAdTrack-CMV中,测序结果与预期一致。酶切鉴定显示由于XbaI位点毗邻碱基的不同导致甲基化的XbaⅠ位点在克隆菌株中不能被有效切开。结论成功构建GM-CSF基因重组腺病毒穿梭载体,XbaⅠ位点对甲基化敏感。
Objective To construct recombinant GM-CSF adenoviral shuttle plasmid and discuss the application of the XbaⅠ enzymatic site, provide the basis of further constructing the recombinant GM-CSF adenovirus and making use of the XbaⅠ multiply cloning site. Methods The mouse spleen cells were separated in vitro and stimulated by ConA. The GM-CSF gene was acquired by RT-PCR with two afterward primers which were different beside the XbaⅠ enzymatic site. After the interest gene was digested by XhoⅠ and XbaⅠ, it was cloned into the pAdTrack-CMV adenoviral shuttle plasmid. The efficiency of the two recombinant plasmids enzymatic identification was compared and verified by sequencing. Results The mouse GM-CSF gene was acquired successfully by RT-PCR and cloned into pAdTrack-CMV adenoviral shuttle plasmid. The sequencing results was identical with the anticipation, and the result of enzyme identification displayed that because of the different bases beside the XbaⅠ site, it could not work well in the cloning bacteria. Conclusion The recombinant GM-CSF adenoviral shuttle plasmid was successfully constructed, and the XbaⅠ site is sensitive to the Dam methylation.
出处
《解剖学研究》
CAS
2006年第3期176-179,共4页
Anatomy Research
关键词
粒细胞-巨噬细胞集落刺激因子
重组腺病毒
甲基化
Granulocyte-macrophage colony-stimulating factor（GM-CSF）
Recombinant adenovirus
Methylation
