CAJ | 학술논문

目的：构建携带小鼠促血管生成素-1（ Ang-1）基因的重组腺病毒载体并鉴定。方法化学合成小鼠 Ang-1基因经PCR扩增后亚克隆至穿梭质粒，重组质粒转化感受态细胞，所得转化子经PCR电泳分析并测序鉴定，携带外源基因的腺病毒穿梭质粒与携带腺病毒大部分基因组的辅助包装质粒共转染人胚肾细胞系HEK 293细胞，反复冻融获取重组腺病毒，终点稀释法测定重组腺病毒滴度，所得腺病毒转染293T细胞48 h收集培养上清，通过 Western Blot分析Ang-1蛋白的表达。结果 PCR产物电泳分析可见大小约1．5 kbp的特征性条带，测序结果显示基因序列与GenBank提供的Ang-1序列一致，经HEK293细胞包装后腺病毒滴度为1×109 pfu／ml，转染293T细胞后培养上清Western Blot分析可见大小58 kD特征性条带，即转染后能分泌正确大小的重组Ang-1蛋白。结论 Ang-1基因重组腺病毒载体可成功构建，为进一步研究Ang-1蛋白对造血干细胞动员及血管新生的作用提供实验依据。
목적：구건휴대소서촉혈관생성소-1（ Ang-1）기인적중조선병독재체병감정。방법화학합성소서 Ang-1기인경PCR확증후아극륭지천사질립，중조질립전화감수태세포，소득전화자경PCR전영분석병측서감정，휴대외원기인적선병독천사질립여휴대선병독대부분기인조적보조포장질립공전염인배신세포계HEK 293세포，반복동융획취중조선병독，종점희석법측정중조선병독적도，소득선병독전염293T세포48 h수집배양상청，통과 Western Blot분석Ang-1단백적표체。결과 PCR산물전영분석가견대소약1．5 kbp적특정성조대，측서결과현시기인서렬여GenBank제공적Ang-1서렬일치，경HEK293세포포장후선병독적도위1×109 pfu／ml，전염293T세포후배양상청Western Blot분석가견대소58 kD특정성조대，즉전염후능분비정학대소적중조Ang-1단백。결론 Ang-1기인중조선병독재체가성공구건，위진일보연구Ang-1단백대조혈간세포동원급혈관신생적작용제공실험의거。
Objective To construct and identify recombinant adenovirus vectors carrying mouse angiopoietin -1(Ang-1) gene. Methods The Ang-1 gene sequence was made by chemosynthesis and amplified by PCR ,and then subcloned into shuttle plasmid .The recombinant plasmid was transformed into competent cell , the transformants was identified by PCR electrophoresis analysis and DNA sequencing .The adenovirus shuttle plasmid carrying exogenous gene and auxiliary packaging plasmid carrying most of the adenovirus genome were cotransfected into HEK293 cells.The recombinant adenovirus was obtained by repeated freezing and thawing.The recombinant adenovirus titer was detected by endpoint dilution assay .The 293T cells were transfected with the recombinant adenovirus ,and then the supernatants were harvested following the 48-hour transfection for a Western Blot analysis on the expression of Ang-1 protein.Results The PCR electrophoresis analysis showed that there was a characteristic band (approximately 1.5 kbp in size).The DNA sequencing showed that the Ang-1 gene sequence was in accordance with the GenBank sequence .The recombinant adenovirus titer was about 1 ×109 pfu/ml after HEK293 packaging.The Western Blot analysis on the supernatants following 293T cell transfection showed that there was a characteristic band of 58 kD,indicating the recombinant Ang-1 protein of the correct size was obtained after transfection .Conclusion The recombinant adenovirus vector carrying Ang-1 gene has been constructed successfully ,which could provide the experimental evidences for further research on the effect of Ang-1 protein on hematopoietic stem cell mobilization and angiogenesis .