CAJ | 학술논문

目的制备用于靶向肿瘤血管中的凝血血浆蛋白的CREKA/tTF融合蛋白,并鉴定其生物学活性.方法利用PCR技术构建CREKA与tTF的融合基因,克隆至表达载体pET22b(+),在E.coli BL21中表达,镍亲和色谱柱纯化,梯度透析复性.利用凝血时间,结合荧光定量测定等实验在体外鉴定该融合蛋白的活性.结果获得序列正确的CREKA/tTF/pET22b(+)重组子,融合蛋白在E.coli BL21中高效表达.纯化后的融合蛋白具有引起血液凝固的功能,且能与凝血血浆蛋白结合.结论成功构建CREKA/tTF/pET22b(+)重组子,CREKA/tTF融合蛋白具有TF凝血活性及CREKA的结合活性.
목적 제비용우파향종류혈관중적응혈혈장단백적CREKA/tTF융합단백,병감정기생물학활성.방법 이용PCR기술구건CREKA여tTF적융합기인,극륭지표체재체pET22b(+),재E.coli BL21중표체,얼친화색보주순화,제도투석복성.이용응혈시간,결합형광정량측정등실험재체외감정해융합단백적활성.결과 획득서렬정학적CREKA/tTF/pET22b(+)중조자,융합단백재E.coli BL21중고효표체.순화후적융합단백구유인기혈액응고적공능,차능여응혈혈장단백결합.결론 성공구건CREKA/tTF/pET22b(+)중조자,CREKA/tTF융합단백구유TF응혈활성급CREKA적결합활성.
Purpose To prepare a novel fusion protein of CREKA and tTF as a universal carrier targeting to cancer,and to analyze its activities.Methods CREKA and tTF gene were acquired by PCR,and inserted into plasmid pET22b(+)to construct recombinant plasmid CREKA/tTF/pET22b(+),and the fusion gene was expressed in E.coli BL21.The fusion protein Wag purified through Nickel-affinity chromatography column.After purifying,the fusion protein was refold by subsequent dialysis.The activities of the fusion proteins were measured by coagulation timing and quantitative fluorescence test in vitro.Results The recombinant plasmid CREKA/tTF/pET22b(+)with correct sequence was obtained.The fusion protein was highly expressed in E.coli BL21.The coagulation of the fusion protein Was determined by the coagulation test.And the capability of the fusion protein effectively binding to clotted plasma proteins is identified in quantitative fluorescence test.Conclusion The recombinant plasmid CREKA/tTF/pET22b(+)with correct sequence was built.The fusion protein CREKA/tTF with both TF and CREKA activity was successfully obtained.