中文摘要：

以人凝血因子Ⅶ（FⅦ） cDNA和编码力达霉素（LDM）辅基蛋白LDP的质粒（pET-VH-LDP）为模板,通过PCR及搭桥PCR构建原核表达人凝血因子Ⅶ轻链（the light chain of FⅦ,lhFⅦ）和LDP融合蛋白lhFⅦ-LDP的重组质粒pET19b-lhFⅦ-LDP.重组质粒转化感受态表达菌株E.coli Rosetta-gami B（DE3） pLysS,诱导表达后亲和纯化,并以Westernblot鉴定得到的融合蛋白lhFⅦ-LDP.将lhFⅦ-LDP与LDM的烯二炔发色团（AE）进行分子组装制备强化融合蛋白药物lhFⅦLDM.用免疫共沉淀（co immunoprecipitation,co IP）检测lhFⅦ-LDP与人肺腺癌细胞A549组织因子（TF）的结合特异性,并通过裸鼠移植瘤实验研究lhFⅦ-LDM对A549细胞体内致瘤性的影响.结果显示,lhFⅦ-LDP能够与TF特异性结合,并且,强化融合蛋白lhFⅦ-LDM能够显著抑制A549细胞的体内致瘤性.

英文摘要：

Standard and bridge PCR were employed, using human coagulation factor Ⅶ （FⅦ） cDNA and lidamycin （LDM） apoprotein LDP-coding plasmid （pET-VH-LDP） as templates, to construct the recombinant plasmid pET19b-lhFⅦ-LDP for prokaryotic production for the fusion protein lhFⅦ-LDP, which contains human coagulation factor Ⅶ light chain （light chain of F Ⅶ, lhF Ⅶ） at N-terminal and LDP domain at C-terminal. The recombinant plasmid was transformed into competent E. coli RosettagamiB（DE3） pLysS. The recombinant protein was induced and purified by affinity chromatography, followed by a confirmation with Western blot. The purified recombinant protein was then assembled with enediyne chromophore of LDM （AE） to produce the enhanced recombinant protein drug lhFⅦ-LDM. Specific interplay between lhFⅦ-LDM and tissue factor （TF） on the surface of A549 cells was tested by co-immunooprecipitaiton. Through the xenografts experiments we can analysis the effects of lhFⅦ- LDM on tumorigenicity of A549 cells in nude mice. The results showed that energized fusion protein lhFⅦ-LDM can significant inhibit tumorigenicity of A549 in vivo by recognizing TF of solid tumors.