人神经菌毛素-1基因重组腺病毒的构建及鉴定

Construction and identification of recombinant adenovirus vector of human neuropilin-1 gene

目的构建含有人神经菌毛素1（NRP-1）基因和3Flag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pDC3153Flag载体片段连接，构建穿梭质粒pDC315-NRP-1-3Flag；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pDC315NRP-1-3Flag的构建；再通过荧光显微镜和Westernblot方法，分别检测质粒pDC315-NRP1-3F1ag和重组腺病毒转染HEK293细胞后NRP1蛋白的表达情况。结果经PCR和测序分析，证实穿梭质粒pDC315-NRP-1-3F1ag与设计一致；穿梭质粒pDC315-NRP13Flag和重组腺病毒分别转染HEK293细胞后，经WesternBlot均检测出在95-130ku处有条特征带，其大小和NRP-1-3F1ag融合蛋白（104ku）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。

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 Age and Nhe restriction endonuclease. Then the DNA was ligated into linearized pDC315-3Flag vector. After having been constructed, the pDC315-NRP-1-3Flag plasmid was co-transfected with framework plasmid pBHGloxAE1, 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 Cytopathic effect was observed by inverted pDC315-NRP-1-3FIag. 95-130 ku was detect shuttle plasmid pDC315-NRP-1-3Flag and design were consistent. phase contrast after transfecting HEK293 cells with shuttle plasmid ed 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 （104 ku）, with a titer of 2.00E--llPFU/mL. Ooflclusion The recombinant adenovirus vector for gene was successfully constructed expressed in HEK 293 cells. fusion protein human NRP-1