CAJ | 학술논문

目的：构建含有人神经菌毛素-1(NRP-1)基因和3 Flag标签蛋白基因的腺病毒载体，为研究该基因对肿瘤细胞与其周围间质细胞的相互作用的影响提供实验基础。方法应用AgeⅠ和NheⅠ限制性内切酶酶切含有人 NRP-1基因的质粒，再将其与酶切线性化的 pDC315-3 Flag 载体片段连接，构建穿梭质粒 pDC315-NRP-1-3 Flag；应用腺病毒骨架质粒 pBHGlox△E13Cre与此穿梭质粒共转染 HEK293细胞，产生重组腺病毒，进而对重组腺病毒进行扩增、纯化及滴度测定；用PCR和基因测序方法验证穿梭质粒 pDC315-NRP-1-3Flag 的构建；再通过荧光显微镜和 Western blot方法，分别检测质粒 pDC315-NRP-1-3Flag和重组腺病毒转染 HEK293细胞后 NRP-1蛋白的表达情况。结果经PCR和测序分析，证实穿梭质粒 pDC315-NRP-1-3 Flag与设计一致；穿梭质粒 pDC315-NRP-1-3 Flag 和重组腺病毒分别转染 HEK293细胞后，经 Western Blot均检测出在95~130 ku处有条特征带，其大小和 NRP-1-3Flag 融合蛋白(104 ku)相吻合；滴度测定为2.00E+11PFU/mL。结论成功构建了人 NRP-1基因重组腺病毒载体，并能在HEK293细胞中表达。
목적：구건함유인신경균모소-1(NRP-1)기인화3 Flag표첨단백기인적선병독재체，위연구해기인대종류세포여기주위간질세포적상호작용적영향제공실험기출。방법응용AgeⅠ화NheⅠ한제성내절매매절함유인 NRP-1기인적질립，재장기여매절선성화적 pDC315-3 Flag 재체편단련접，구건천사질립 pDC315-NRP-1-3 Flag；응용선병독골가질립 pBHGlox△E13Cre여차천사질립공전염 HEK293세포，산생중조선병독，진이대중조선병독진행확증、순화급적도측정；용PCR화기인측서방법험증천사질립 pDC315-NRP-1-3Flag 적구건；재통과형광현미경화 Western blot방법，분별검측질립 pDC315-NRP-1-3Flag화중조선병독전염 HEK293세포후 NRP-1단백적표체정황。결과경PCR화측서분석，증실천사질립 pDC315-NRP-1-3 Flag여설계일치；천사질립 pDC315-NRP-1-3 Flag 화중조선병독분별전염 HEK293세포후，경 Western Blot균검측출재95~130 ku처유조특정대，기대소화 NRP-1-3Flag 융합단백(104 ku)상문합；적도측정위2.00E+11PFU/mL。결론성공구건료인 NRP-1기인중조선병독재체，병능재HEK293세포중표체。
Objective To construct a adenovirus vector containing human NRP-1 gene and 3Flag gene to interaction between tumor and interstitial cell.Methods Plasmid containing NRP-1 gene was digested by AgeⅠand NheⅠ restriction endonuclease.Then the DNA was ligated into linearized pDC315-3Flag vector.After having been constructed,the pDC3 1 5-NRP-1-3 Flag plasmid was co-transfected with framework plasmid pBHGlox△E1 , 3Cre into HEK 293 cells to obtain the homologous recombinant adenovirus,which was then amplified and purified its titer tested.Expression of NRP-1 protein was detected using Western blot.Results Polymerase chain reaction and sequencing analysis confirmed that the shuttle plasmid pDC3 1 5-NRP-1-3 Flag and design were consistent. Cytopathic effect was observed by inverted phase contrast after transfecting HEK2 9 3 cells with shuttle plasmid pDC315-NRP-1-3Flag.95-130 ku was detected by Western lot after transfecting HEK293 cells with shuttle plasmid pDC315-NRP-1-3Flag and recombinant adenovirus,the size being consistent with the NRP-1-3Flag fusion protein (104 ku),with a titer of 2.00E+11PFU/mL.Conclusion The recombinant adenovirus vector for human NRP-1 gene was successfully constructed expressed in HEK 2 9 3 cells.