摘要
目的 构建VEGFR 2胞外段基因真核表达质粒,并在体外进行表达和鉴定。方法 采用PCR技术扩增VEGFR 2分子胞外段编码基因FLK12 6 5- 2 4 93,先将该片段插入到pMDT 18载体中,再亚克隆至真核表达载体pSG .SS .YL ,构建VEGFR 2分子胞外段编码基因表达质粒。经限制性酶切鉴定和DNA序列测定结果证实后,将重组质粒pSG .SS .FLK12 6 5- 2 4 93.YL转染COS 7细胞,检测其体外表达。结果 免疫印迹和免疫细胞化学分析结果分别表明转染细胞上清液和胞浆内均有目的分子的存在。结论 构建的DNA疫苗可在真核细胞内正确表达,这为进一步的动物实验打下了基础。
Objective To construct the eukaryotic expression vector for VEGFR-2 extracellular domain gene and express it in vitro. Methods The fragment of VEGFR-2 extracellular domain was amplified by PCR and inserted into pMDT-18 plasmid and then subcloned into eukaryotic expression vector pSG.SS.YL. The recombinant plasmid pSG.SS.FLK1 265-2 493 .YL was transfected into COS-7 cell and the expressed product was detected by Westernblot timg and immunocytochemistry. Results Both Western blotting and immunocytochemistry demons- trated that the VEGFR-2 extracellular domain existed in the cultural supernatant and cytoplasmic of COS-7 cells tansfected by recombinant plasmid. Conclusion The constructed DNA vaccine can be expressed in vitro, which may pave a way for further studies in animals.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2005年第2期96-99,共4页
Immunological Journal
