腺病毒介导的小鼠可溶性endoglin和可溶性血管内皮生长因子受体-1重组蛋白的构建和功能初步鉴定

Construction and function identification of recombinant mouse soluble endoglin and soluble fms-like tyrosine kinase-1 mediated by adenovirus

目的构建腺病毒介导的小鼠可溶性endoglin（sEng）和可溶性血管内皮生长因子受体-1（sFlt-1）重组蛋白，观察其对内皮细胞管化及滋养细胞迁移的影响。方法提取小鼠胎盘组织总RNA，通过RT—PCR获得sEng和sFlt-1 cDNA，插入到穿梭载体pAdTrack—CMV中，获得pATC-sEng和pATC—sFh-1质粒，转化其至pAdeasy-1受体菌，选阳性克隆转染293A细胞，获得Ad／sEng和Ad／sFlt-1，制备高滴度病毒液。用病毒感染COS7细胞，检测sEng和sFlt-1蛋白表达，并通过成管和划痕实验观察两种蛋白对人脐静脉血管内皮细胞（HUVEC）管化和Bewo细胞迁移的影响。结果成功获得sEng和sFlt-1重组腺病毒，其滴度分别为2．2×10^11pfu／ml和2．0X10^11pfu／ml。感染COS7细胞后，sEng和sFlt-1蛋白均较高表达，并能抑制HUVEC管化及Bewo细胞迁移，两者有协同作用。结论构建的腺病毒介导的小鼠sEng和sFlt-1重组蛋白对血管生成和滋养细胞迁移有明显抑制作用。

Objective To construct the recombinant adenovirus Ad/sEng and Ad/sFlt-1, and investigate their effects on angiogenesis and the migration of human umbilical vein endothelial cells （HUVEC） and trophoblast. Methods The sEng and sFlt-1 cDNA were obtained from mouse placenta by RT-PCR, which were inserted into the shuttle plasmid pAdTrack-CMV. After confirmation by endonuclease and sequencing, we transferred them into Escherichia coll. BJ5183- AD-1 cells were applied to obtain the homologous recombinants, which were transfected into 293A cells to obtain Ad/sEng and Ad/sFlt-1, and COS7 cells were infected with them. The expressions of sFlt-I and sEng were detected by Western blot. The effects of both proteins on HUVEC tube formation and BeWo migration were investigated. Results High titers of Ad/sEng and Ad/sFlt-1 were obtained （2.2 ×10^11 pfu /ml and 2. 0× 10^11 pfu/ml, respectively） and both sFlt1 and sEng proteins were efficiently expressed, which inhibited the HUVEC tube formation and BeWo cell migration. Conclusions Adenovirus mediated mouse sFlt-1 and sEng has been constructed and expressed successfully in vitro with inhibitory effects on angiogenesis and the migration of HUVEC and trophoblast.