强化融合蛋白lhFⅦ-LDM对人肺腺癌细胞A549体内致瘤性的抑制  

The inhibitory effect of energized fusion protein lhFⅦ-LDM on the tumorigenicity of A549 cells in vivo

作　　者：廖东升[1] 阳宇[1] 陈金武[1] 马登佼 李灵[1] 

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,成都610064

出　　处：《四川大学学报（自然科学版）》2014年第5期1029-1034,共6页Journal of Sichuan University(Natural Science Edition)

基　　金：重大新药创制科技重大专项(2009ZX09103-698);国家自然科学基金(31000579);四川省科技创新研究团队专项计划(2011JTD0026)

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(pETVH-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Western blot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lhFⅦ-LDM.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.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.

关 键 词：凝血因子Ⅶ轻链(lhFⅦ) LDM 组织因子(TF) A549细胞 体内致瘤性 

分 类 号：Q25[生物学—细胞生物学]