摘要
目的构建人血管生成素2（Angiopoietin2）原核表达载体。方法体外培养人脐静脉内皮细胞（HUVECs），通过逆转录-聚合酶链反应（RT—PCR）扩增获取Angiopoietin2基因片段，然后将其克隆人pET32a质粒载体表达系统，实现Angiopoietin2在大肠杆菌BL21中的稳定表达，并通过凝胶电泳、SDS．PAGE及基因测序等对表达蛋白进行鉴定。结果从HUVEC中克隆出约1．5kb的Angiopoietin2基因，通过pET32a载体系统实现了Angiopoietin2蛋白的表达，SDS—PAGE、电泳及测序分析证实表达成功，融合蛋白相对分子质量为70×10^3。结论应用基因重组技术成功构建了人Angiopoietin2原核表达载体。
Objective To construct the human Angiopoietin2 prokaryotic expression vector. Methods Complete Angiopoietin2 gene was cloned by reverse transcfiption-polymerase chain reaction （RT-1-CR） from in vitro cultured human umbilical vein endothelial cells （ HUVECs）, then the gene was cloned into pET32a plasmid expression system, and ultimately Angiopoietin2 was stablely expressed in E. coli BL21. Angiopoietin2 protein was then purified and identified by condensate gel electrophoresis, SDS-PAGE and gene sequencing. Results The cloned Angiopoietin2 gene fragment from HUVECs was about 1.5 Kb, the Angiopoietin2 protein was expressed by pET32a plasmid expression system, and it was predicated about 70 kDa protein by SDS-PAGE. Conclusion The prokaryotic expression vector of Angiopoietin2 is successfully constructed by recombinant DNA techniques.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2012年第6期1089-1091,共3页
Chinese Journal of Experimental Surgery
基金
基金项目：中央高校基本科研业务费专项资金资助项目（303275892）
关键词
血管生成素
原核表达载体
Angiopoietin
Prokaryotic expression vector
