摘要
该文主要研究了强化融合蛋白lhFⅦ-LDM对乳腺癌MDA-MB-231细胞的抑制作用。通过PCR和重叠PCR的方法构建了pET19b-lhFⅦ-LDP表达载体,重组质粒转化BL21,经IPTG诱导后表达并用Co^(2+)亲和层析纯化lhFⅦ-LDP融合蛋白,Western blot检测融合蛋白的正确性,再通过分子重组的方法将lhFⅦ-LDP与力达霉素(LDM)的活性发色团(AE)组装成为强化融合蛋白lhFⅦ-LDM。通过免疫共沉淀实验鉴定lhFⅦ-LDP与组织因子(TF)的特异性结合作用,利用平板克隆形成实验观察lhFⅦ-LDM对细胞增殖的影响,采用Hoechst33342染色检测lhFⅦ-LDM诱导MDA-MB-231细胞凋亡情况,建立了人乳腺癌裸鼠肿瘤模型,研究lhFⅦ-LDM对MDA-MB-231肿瘤生长的抑制作用。结果显示,lhFⅦ-LDM强化融合蛋白在体外能很好的诱导MDA-MB-231细胞凋亡,动物实验结果表明,强化融合蛋白对MDA-MB-231肿瘤的生长具有显著的抑制作用。
The aim of this text is to study the inhibitory effect of energized fusion protein IhFVII-LDM on MDA-MB-231 cell. The prokaryotic expression vector pET19b-lhFVII-LDP, composed of a light chain of human factor VII （lhFVII） and an apoprotein of LDM （LDP）, was constructed through normal PCR and overlapping extension PCR. The recombinant vector was further transformed into Escherichia coli strain BL21 for expression under the induction of IPTG. Fusion protein was purified by Co2＋ affinity chromatography. The identity of IhFVII- LDP was confirmed by Western blot assay and energized fusion protein lhFVII-LDM, composed of lhFVII-LDP and the active enediyne （AE）, was obtained by molecular reconstitution. The specific binding activity of IhFVII-LDP with TF was verified by co-immunoprecipitation assay. Cloning form assay was used to detected the inhibitory effect of energized fusion protein on the proliferation of MDA-MB-231 cell. Apoptosis induced by IhFVII-LDM was detected by Hoechst33342. Moreover, nude mice model transplanted with human breast tumor was established to study the inhibitory effect of lhFVII-LDM on breast tumor. The results suggested that lhFVII-LDM could induce MDA-MB-231 cells apoptosis in vitro. The result of animal experiment showed that lhFVII-LDM had significant inhibitory effect on the growth of MDA-MB-231 tumor.
出处
《中国细胞生物学学报》
CAS
CSCD
2011年第9期976-982,共7页
Chinese Journal of Cell Biology
基金
<重大新药创制>国家科技重大专项课题基金(No 2009ZX09103)资助项目~~
