摘要
目的构建分泌型人内皮抑素(ES)的真核表达载体pEGFP-N1-Endostatin重组质粒,鉴定其蛋白的体外瞬时表达。方法以pcDNA3-Endo质粒为模板,通过PCR扩增获得人ES基因片段,且在基因前加信号肽序列,将其定向插入真核表达载体pEGFP-N1中,获得重组质粒pEGFP-N1-ES。采用HindⅢ和BamHⅠ双酶切法、PCR法及插入片段序列测定法鉴定该质粒。利用阳离子脂质体介导法,将其转染到人胚胎肾HEK-293细胞中,采用免疫组织化学法和Westernblot法检测转染细胞内及培养上清液中ES蛋白的表达。结果通过HindⅢ和BamHⅠ双酶切、PCR及测序鉴定证明构建出了含ES基因的真核表达载体,且插入片段正确。采用免疫组织化学法和Western blot法检测表明HEK-293细胞内及培养上清中存在相对分子质量为20 000的人ES蛋白表达。结论成功构建了重组质粒pEGFP-N1-ES真核表达载体,转染HEK-293细胞后可稳定有效地分泌人ES蛋白。
Objective Animal experiment and clinical trials have showed that endostatin （ES） can restrain intraoeular neovessels by intraocular injection. The short half-life of ES and protein correct refolding and preparation process make it difficult to achieve the clinical long-term and high-dose drug requests. Present study was to construct the eukaryotie expression vector pEGFP-N1-Endostatin containing human ES gene in human embryo kidney （HEK-293） cells and detect its transient protein expression in vitro. Methods Recombinant plasmid pEGFP-N1-Endostatin was constructed by which eDNA sequence of ES used as template to gain ES gene fragment and synthesize the upper primer which contains the signal sequence by polymerase chain reaction and was inserted into eukaryotic expression vector pEGFP-N1. The vector was evaluated by Hind III and BamH I double enzyme incision, polymerase chain reaction and sequence analysis. Recombinant plasmid pEGFP-N1-Endostatin was transfeeted into HEK-293 cells by using cationic liposome,immunoeytochemical staining,and Western blot was used to detect the transient expression of human ES protein in the supernatant of transfected cells in vitro. Results The inserted fragment was proved to be correct by HindIII and BamH I double enzyme ineision,polymerase chain reaction and sequence analysis. The size of PCR amplified product of ES and recombined plasmid pEGFP-NI-Endostatin were consistent with the expected figment. Compared with the sequence published in Genebank,the sequence of insert designation was coincident with human ES gene. The immunochemistry determined that transfected HEK-293 ceils showed the brown-yellow staining in cytoplasm. Western blot results indicated that ES protein was expressed in HEK-293 cell supernatant with the relative molecular weight 20 000. Conclusion A eukaryotic expression vector of recombined plasmid pEGFP-N1-Endostatin is successfully constructed and human ES protein is effectively secreted in HEK-293 cells transfected ES gene.
出处
《眼科研究》
CAS
CSCD
北大核心
2009年第10期864-869,共6页
Chinese Ophthalmic Research
基金
贵州省科技厅自然基金资助(黔科合J字[2005]2061)
