CAJ | 학술논문

目的建立人fg12凝血酶原酶纤维蛋白原相关结构域(fibrinogen-related domain,FRED)的原核表达系统,并鉴定表达蛋白的凝血活性.方法应用RT-PCR从外周血单个核细胞扩增fg12凝血酶原酶FRED基因,克隆到原核表达载体pET22b(+),以异丙基-β-D-硫代半乳糖苷诱导重组蛋白表达,重组蛋白经SDS-PAGE电泳和免疫印迹鉴定并用His-Tag柱纯化,以促凝活性(procoagulant activity,PCA)检测鉴定其凝血功能.结果扩增了人fg12凝血酶原酶FRED基因,成功构建了重组pET-FRED原核表达质粒,表达的融合蛋白具有直接促凝血活性.结论建立了人fg12凝血酶原酶FRED的高效原核表达系统,FRED可能为fg12凝血酶原酶促凝血的活性区域.
목적 건립인fg12응혈매원매섬유단백원상관결구역(fibrinogen-related domain,FRED)적원핵표체계통,병감정표체단백적응혈활성.방법 응용RT-PCR종외주혈단개핵세포확증fg12응혈매원매FRED기인,극륭도원핵표체재체pET22b(+),이이병기-β-D-류대반유당감유도중조단백표체,중조단백경SDS-PAGE전영화면역인적감정병용His-Tag주순화,이촉응활성(procoagulant activity,PCA)검측감정기응혈공능.결과 확증료인fg12응혈매원매FRED기인,성공구건료중조pET-FRED원핵표체질립,표체적융합단백구유직접촉응혈활성.결론 건립료인fg12응혈매원매FRED적고효원핵표체계통,FRED가능위fg12응혈매원매촉응혈적활성구역.
Objective To establish a prokaryotic expression system of fibrinogen-related domain(FRED)gene of fgI2 pro-thrombinsae and identify the procoagulant activity of recombinant protein. Methods FRED cDNA was amplified by RT-PCR from peripheral blood mononuclear cells,then cloned into prokaryotic expression vector pET22b( + ). The recombinant plasmid was transferred into E. coli BL21. IPTG was used to induce the expression of recombinant protein,and the recombinant protein was identified by SDS-PAGE eletrophoresis and immunoblot, and purified with his-tag chromatographic column. Coagulation function of the recombinant protein was confirmed by procoagulant activity assay. Results The recombinant plasmid pET-FRED was constructed successfully. The recombinant protein could activate coagulation directly. Conclusion A highly efficient prokaryotic expression system for FRED of fgl2 prothrombinsae was established. FRED may be a domain with activating coagulation of fgl2 prothrombinsae.