摘要
目的：构建小鼠 noggin 基因的重组腺病毒载体并鉴定。方法：采用基因工程技术。经过2次亚克隆将 noggin 基因片段克隆至穿梭质粒 AdTrack-CMV 上,利用 pAdEasy-1系统进行细菌内同源重组后,脂质体转染 293T 细胞包装、扩增。采用 PCR 方法对重组体腺病毒进行鉴定,利用穿梭质粒中带有绿色荧光蛋白 GFP 报告基因,对病毒滴度和感染效率进行监测。结果：酶切鉴定及 PCR 结果证明 noggin 基因重组腺病毒载体构建成功,病毒滴度达6．3×10^(10)pfu/ml。结论：应用细菌内同源重组法成功构建了含小鼠 noggin 基因的重组腺病毒载体。
Objective： To construct the recombinant adenovirus of mouse noggin gene by the homologous recombination in bacteria. Methods： The noggin gene fragment was inserted into the shuttle plasmid pAdTrack-CMV and then homologous recombination was performed with pAdEasy- 1 system in bacteria. The recombinant adenovirus was transfected into 293T cells by lipofectine DOTAP. The target gene was detected by polymerase chain react,ion （PCR）; the titer and its infection rate were determined through detection of the green fluorescent protein （GFP） expression in the shuttle plasmid. Results： Restriction endonuclease and PCR analysis confirmed that the noggin gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 6.3 ×10^10 pfu/ml. Conclusion： The recombinant adenovirus containing noggin gene has been successfully constructed by ho mologous recombination in bacteria.
出处
《解剖学杂志》
CAS
CSCD
北大核心
2006年第1期51-54,共4页
Chinese Journal of Anatomy
基金
国家自然科学基金(30400100)
