FOXL2基因重组腺病毒载体的构建及鉴定

为了构建叉头框L2(Forkhead box L2,FOXL2)基因与绿色荧光蛋白(GFP)基因复制缺陷型腺病毒载体,试验将克隆的FOXL2基因与IRES-GFP片段通过酶切、纯化等方法共同连接到pShuttle-CMV载体中,再与pAdEasy-1腺病毒质粒在BJ5183大肠杆菌中进行同源重组,得到复制缺陷型AD-FOXL2腺病毒载体,再用PacⅠ酶线性化后转染HEK293细胞,观察绿色荧光表达,并测量病毒效价。结果表明:将AD-FOXL2腺病毒载体用PacⅠ酶线性化后,得到1个大于23 kb的大片段和1个4.5 kb的特异性小片段,证明同源重组成功;将所得的腺病毒载体转染HEK293细胞,可以观察到GFP的表达,证明包装成功,并测得病毒效价为1×10-8.61/0.1 mLTCID50。

Construction and identification of the recombinant adenovirus vectors for FOXL2 gene

To construct recombinant adenovirus plasmid expressing FOXL2 gene and GFP,the experiment adopted the plasmid pCMV5-FOXL2 which was double-digested,purified and pluged into the pShuttle-CMV vector with IRES-GFP fragment.After sequenced,the recombinant adenovirus vector Ad-FOXL2 was constructed with FOXL2 and GFP through homologous recombination technique in E.coli of BJ5183 using Adeasy-1 system.After linearized by PacⅠ,the vector was transfected into HEK293 cell and then the viral titer was checked by GFP.The results indicated that the recombinant adenovirus vector digested by restrictive endonuclease PacⅠwas constructed successfully.It could be cut into two fragments,one is more than 23 kb,and 4.5 kb another.Twenty-four hours after transduction mediated by liposome,the fluorescence was observed in HEK293 cells.The adenovirus titer reached TCID50=1×10-8.61/0.1 mL,which was checked by GFP.This revealed that the recombinant adenovirus vector containing FOXL2 gene was successfully constructed.It lays the foundation for further study on expression and purification of FOXL2 as well as the mechanisms of sex control.