血管内皮细胞生长因子受体短发夹环RNA真核表达载体的构建  

The Generation of Eukaryotic Expression Vector of ShRNA Specific For VEGF Receptors Flt-1 and KDR

作　　者：许文林[1] 沈慧玲[1] 袁伟[2] 江云伟[2] 阮长耿[1] 

机构地区：[1]苏州大学附属第一医院江苏省血液研究所,江苏苏州215006 [2]镇江市第一人民医院,江苏镇江212002

出　　处：《苏州大学学报（医学版）》2004年第4期479-483,共5页Suzhou University Journal of Medical Science

摘　　要：目的　构建血管内皮细胞生长因子受体 (VEGFR)Flt 1和KDR特异性短发夹环RNA(ShRNA)真核表达载体。方法　采用人U6SnRNA启动子 ,分别把被 9bp序列间隔的 19bp长短的Flt 1和KDR靶序列的反向重复序列 ,置于 pEGFP C1质粒载体中 ,分别构建成Flt 1和KDR短发夹环RNA ,产生重组质粒 pEGFP C1/U6/Flt 1及pEGFP C1/U6/KDR。分别用Pst和SalⅠ酶切鉴定 ;并将经酶切鉴定后的重组质粒作测序分析。结果　VEGF受体Flt 1和KDR的ShRNA片段被成功克隆进 pEGFP C1质粒中 ,SalⅠ酶切鉴定可切出 4 0 0bp大小的片段 ,DNA测序分析显示 ,重组质粒ShRNA编码序列与设计片断的序列完全一致。结论　针对Flt 1和KDR的特异性ShRNA真核表达载体PEGF/U6/Flt 1及PEGF/U6/KDR的成功构建 。Objective To generate eukaryotic expression vector of short hairpin RNA(ShRNA) specific for VEGF receptors Flt-1 and KDR. Methods A plasmid pEGFP-C1 containing human U 6 snRNA promoter was ligated to a 19 bp reverse repeated motif of Flt-1 and KDR targer sequeace with 9 bp spacer,respectively.Enzyme digestion and DNA sequencing were used to examine whether the recombinant plasmid pEGFP-C1/U 6/Flt-1 and pEGFP-C1/U 6/KDR were correct. Results The Flt-1-shRNA and KDR-shRNA expression frame were successfully inserted into eukaryotic expression vector pEGFP-C1 with U 6 suRNA promoter and termination signal of RNA polymerase Ⅲ.Enzyme digestion by Pst Ⅰ and Sall showed a 400bp DNA fragment.The sequences of shRNA recombinant plasmid were the same as that of designed fragments. Conclusion Eukargotic expression vectors of ShRNA specific for Flt-1 and KDR were generated successfully.Our study may start a new approch to research the gene function of VEGF receptors Flt-1 and KDR and the method of gene therapy.

关 键 词：RNA干扰 FLT-1 KDR 短发夹环RNA U6SnRNA启动子 

分 类 号：R394.3[医药卫生—医学遗传学] R733.7[医药卫生—基础医学]