CAJ | 학술논문

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,然后将其克隆入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70×103.结论应用基因重组技术成功构建了人Angiopoietin2原核表达载体.
목적 구건인혈관생성소2( Angiopoietin2)원핵표체재체.방법 체외배양인제정맥내피세포(HUVECs),통과역전록-취합매련반응(RT-PCR)확증획취Angiopoietin2기인편단,연후장기극륭입pET32a질립재체표체계통,실현Angiopoietin2재대장간균BL21중적은정표체,병통과응효전영、SDS-PAGE급기인측서등대표체단백진행감정.결과 종HUVEC중극륭출약1.5kb적Angiopoietin2기인,통과pET32a재체계통실현료Angiopoietin2단백적표체,SDS-PAGE、전영급측서분석증실표체성공,융합단백상대분자질량위70×103.결론 응용기인중조기술성공구건료인Angiopoietin2원핵표체재체.
Objective To construct the human Angiopoietin2 prokaryotic expression vector.Methods Complete Angiopoietin2 gene was cloned by reverse transcription-polymerase chain reaction (RTPCR) fromin 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.