Preparation of Fusion Protein lhFVII-LDM and Its Inhibitory Effect on Breast Tumor

Abstract: The aim of this text is to study the inhibitory effect of energized fusion protein lhFVII-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 lhFVIILDP 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 lhFVIILDP 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 lhFVII-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.

CSTR: 32200.14.cjcb.2011.09.0004