摘要
目的建立人fgl2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fgl2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fgl2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fgl2凝血酶原酶FRED的高效原核表达系统,FRED可能为fgl2凝血酶原酶促凝血的活性区域。
Objective To establish a prokaryotic expression system of fibrinogen-related domain（FRED）gene of fgl2 prothrombinsae 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.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2009年第5期660-663,共4页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
广西壮族自治区自然科学基金资助项目(No.桂科自0447025)
