反义VEGF_(165)真核表达载体的构建和表达

Construction and identification of antisense VEGF_(165) eukaryotic expression vector

目的 进行抗血管生成治疗人脑胶质瘤实验 ,构建和鉴定携带人反义 VEGF1 6 5 基因的真核表达载体 .方法 将VEGF1 6 5 c DNA反向克隆入 pc DNA3,构建 CMV启动子控制的真核表达载体 pc DNA- AVEGF1 6 5 ,用酶切鉴定结果 ;应用该载体转染人脑胶质瘤细胞 SHG44 ,G418筛选阳性克隆 ;Western blot和免疫组化检测转染前、后瘤细胞的 VEGF表达 .检测转染前、后瘤细胞的生物学性状 .结果 获得反向构建的 pc DNA- AVEGF1 6 5 真核表达载体 ,VEGF1 6 5 反义 RNA阻断了 SHG44细胞的 VEGF表达 ,该细胞生物学性状不受外源基因的表达影响 .结论 成功构建的反义 VEGF1 6 5 真核表达载体 。

AIM To construct and to identify eukaryotic expression vector carrying human antisense VEGF 165 cDNA to cure human glioma with antiangiogenesis method. METHODS VEGF 165 cDNA was inserted into polylinker site ofeukaryotic expression vector pcDNA3 to construct pcDNA AVEGF 165 , in which restriction enzyme analysis was used to confirm the reverse orientation of the VEGF 165 cDNA from the individual transformants. The vector was transfected into human gliomas cell and the positive clone was screened by G418. The VEGF expression of SHG44 cells before or after transfection was detected by Western blot and immunohistochemistry. The biological characteristics of SHG44 cells before and after transfection was inspected. RESULTS The pcDNA AVEGF 165 vector was obtained. VEGF 165 expression of SHG44 cells was blocked by antisense VEGF 165 RNA. The characteristics of the tumor cells were not influenced by expression of the antisense gene. CONCLUSION The successful construction of antisense VEGF 165 eukaryotic expression vector is of significance for glioma specific antisense gene therapy.