人Sef基因重组腺病毒载体的构建与鉴定

Construction and Characterization of hSef Recombinant Adenoviral Vectors

构建人Sef-L和Sef-S基因的复制缺陷型重组腺病毒表达载体,为研究Sef的功能和作用机制以及Sef的基因治疗奠定基础。通过PCR方法以hSef的表达质粒为模板扩增得到hSef的编码序列,亚克隆到穿梭载体pAdTrack-CMV中,经测序验证之后,将穿梭载体使用Pine I酶切线性化,然后与腺病毒基因组质粒pAdEasy-1共转化大肠杆菌BJ5183,得到重组的Ad-hSef-L和Ad-hSef-S质粒,最后将Ad-hSef-L和Ad-hSef-S质粒使用Pac I线性化,转染到HEK293细胞中,包装收获病毒颗粒,免疫印迹实验鉴定表达,荧光素酶报告实验验证其功能。成功构建了人Sef基因的复制缺陷型重组腺病毒表达载体,获得了有功能的Ad-hSef-L和Ad-hSef-S病毒重组子。

Sef （similar expression to fgf genes） was identified as a feedback antagonist of FGF signaling in zerbrafish, mouse and human. To construct recombinant adenoviral vectors expressing hSef-L and hSef-S, the coding sequences of the two isoforms were amplified and ligated into pAdTrack-CMV, forming shuttle vectors pAdTrack-CMV/hSef-L-Myc and pAdTrack-CMV/hSef-S-Myc. After sequence confirmation, these two shuttle vector plasmids were linearized by Pme I and then co-transformed respectively with the adenoviral genome vector pAdEasy-1 into E. coli BJ5183. The successful recombinants were selected by Kanamycin and confirmed by Pac I digestion. The recombinant vectors Ad-hSef-L-Myc and Ad-hSef-S-Myc were finally digested with Pac I and transfected into HEIr93 ceils to pack into viral particles. The virus were amplified in 293 cells and used to infect MEF cells. Western blotting analysis was used to demonstrate the expression of hSef-L-Myc and hSef-S-Myc proteins. The inhibitory effects of the adenovirus mediated Sef expression on FGF signaling was further evaluated by Elk luciferase reporter assay. Our results indicated the constructed virus could produce effectively the proteins and then inhibit FGF signaling in MEF cells.