人源分泌型血管内皮细胞生长因子受体flt-1(Ⅰ～Ⅳ/区)的基因克隆及其腺病毒载体的构建和表达

Gene Cloning of Secreted VEGF Receptor flt-1 (Ⅰ～Ⅳ extracellular domain) and The Corresponding Recombinant Adenovirus Construction and Expression

肿瘤的生长、转移与血管形成密切相关 ,利用基因治疗的方法将抗血管形成的因子导入体内是目前肿瘤生物治疗研究的重要策略 .血管内皮细胞生长因子 (VEGF)在血管生成中起重要作用 ,因此阻断VEGF与相应受体的结合成为抗血管形成的重要靶点 .通过RT PCR从人脐静脉内皮细胞克隆了VEGF受体Flt 1的信号肽及胞外Ⅰ～Ⅳ区cDNA ,即可溶性sFlt 1的cDNA片段 .利用Ad Easy体系 ,在细菌BJ5 183中同源重组后 ,转染包装细胞 2 93,成功包装出重组flt 1腺病毒 ,利用它可有效地感染低分化胃黏液腺癌细胞株MGC80 3.经RT PCR ,免疫沉淀及免疫印迹等不同方法检测表明 ,被感染细胞能表达并分泌Flt 1的胞外区蛋白 ,为后续进行抗肿瘤血管形成的基因治疗研究奠定了基础 .

The increasing data show that the soluble form of Flt 1 have the ability to block the biological activity of VEGF due to its high affinity to VEGF and formation of homologous and heterogenous dimmer with transmembrane Flt 1 and KDR. The binding activity of Flt 1 focuses on its Ⅱand Ⅲ extracellular domain. cDNA fragments encoding for signal peptide and flt 1 Ⅰ～Ⅳ extracellular domain were amplified through RT PCR with a pair of specific primers from human umbilical vein endothelial cells. Then, the sflt 1 gene was subcloned into pcDNA3 EF 1 vector, and the expression unit of EF 1 flt 1 was subcloned into shuttle vector of pAdTrack CMV, linearized pAdTrack EF 1 flt 1 was co transformed into BJ5183 cells with adenoviral genomic plasmid of pAdEasy 1. The identified recombinant DNA was transfected into 293 cells to package adenovirus. From the supernatant and cell lysis, the presence of recombinant adenovirus was proved by PCR, and RT PCR detection demonstrated the transcription of flt 1 in MGC803 cells infected with Ad Track EF 1 flt 1, and immunoprecipitation reveals that soluble Flt 1 can be secreted into the culture supernatant of infected MGC803 cells. These results indicated that tumor cells infected with the prepared recombinant adenovirus vector Ad Track EF 1 flt 1 can secrete soluble Flt 1, which will be further used for in vivo antiangiogenesis experiments.