VEGF-C真核表达载体的构建及蛋白表达  

The Construction and Expression of Eukaryotic Expression Vector Harboring Homo Sapiens Vascular Endothelial Growth Factor-C Gene

作　　者：丁凯阳[1] 白霞[1] 董宁征[1] 余自强[1] 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

出　　处：《苏州大学学报（医学版）》2005年第2期234-237,共4页Suzhou University Journal of Medical Science

摘　　要：目的　构建人血管内皮生长因子(VEGF- C)编码序列基因真核表达载体,为探讨VEGF- C的生物学功能奠定基础。方法　根据已公布的序列设计引物,用PCR方法从肿瘤细胞株cDNA中扩增出人VEGF- C编码序列基因片段,测序正确后用限制性内切酶将目的片段插入PcDNA3.1/V5 His TOPO载体中。采用酶切和PCR鉴定后通过脂质体介导转染至CHO细胞中进行瞬时表达。结论　经RT -PCR扩增转染细胞cDNA和Westernblotting检测证实重组质粒pcDNA3.1 VEGF- C能在宿主细胞中高效表达。Objective To study the biological activity of VEGF-C,and to consbuct eukaryotic expression vectors harboring homo sapiens VEGF-C gene was constructed.Methods The human VEGF-C cDNA was amplied from the pancreatic cancer cell line A549 by PCR and the PCR products which was in line with the reported human VEGF-C gene were inserted into eukaryotic expression vectors PcDNA3.1/V5-His-TOPO by enzyme restriction and ligation. Methods The recombinant expression plasmid was transfected into CHO cells and the transient expression product was analyzed by Western blotting. Conclusion The results demonstrated that the eukaryotic expression vector PcDNA3.1/V5-His-TOPO-VEGF-C was successfully constructed and can express its corresponding protein efficiently, which provides a basis for the further study of biological function of human VEGF-C.

关 键 词：蛋白表达 人血管内皮生长因子 基因真核表达载体 RT-PCR扩增 BLOTTING VEGF-C Western 限制性内切酶 编码序列 cDNA 生物学功能 肿瘤细胞株 PCR方法 CHO细胞 脂质体介导 PCR鉴定 设计引物 基因片段 转染细胞 瞬时表达 高效表达 

分 类 号：R735.2[医药卫生—肿瘤] R331.32[医药卫生—临床医学]