Ptd-od-ha Fusion Protein Induced Apoptosis And Inhibited Proliferation In Bcr/abl Positive Cells

Bcr/Abl fusion protein is composed of multiple functional domains. Oligomerization domain(short for OD),located in the N-terminal of Bcr which consist of 1～72 amino acids(aa), mediates Bcr/Abl homologous oligomerization ,which is important for the Bcr/Abl auto-phosphorylation and induces alteration of molecular comformation , finally leads to abnormal activation of Abl tyrosine kinase. Thus we speculate that the tyrosine kinase activity of Bcr/Abl will be inhibited by transduction exogenous synthetic oligomerization domain protein to interrupt oligomerization of Bcr/Abl. This strategy is expected to become a new treatment method for CML. We applied a new efficient protein delivery system: TAT-protein transduction domain to transduct exogenous OD proteins into cells ,adding a HA tag for following immunological detection.We constructed a pPTD-OD-HA expression plasmid which produced the PTD-OD-HA fusion protein in E.coli BL21 (DE3). The fusion protein was purified by Ni+-NTA His·Bind Resin and was transducted into CML cells. Apoptotic and proliferative effects in these cells was detected. These are the basis for animal experiments. The main experiments used in this subject as follows:1. The construction, prokaryotic expression, purification and identification of PTD-OD-HA fusion protein: the OD, HA, and PTD gene fragments were successively cloned into pET32a (+) prokaryotic expression vector. After verifying its correctness through enzyme digestion and sequencing, pPTD-OD-HA recombinant plasmid was transformed into E. coli BL21 (DE3) strain. IPTG induced the expression of PTD-OD-HA fusion protein. After purification by Ni+-NTA His·Bind Resin, the fusion protein was identified by SDS-PAGE coomassie brilliant blue staining and western blot. OD-HA fusion protein was constructed, expressed and purified for control.2. The apoptotic effect PTD-OD-HA fusion protein on Bcr / Abl-positive cells: Apoptosis was detected by flow cytometry, DNA ladder assay and transmission electron microscopy. The changes of bax, bcl-2 in mRNA and protein were detected by RT-PCR and western blot respectively.3. The proliferative effect PTD-OD-HA fusion protein on Bcr/Abl-positive cells: cell growth was detected by MTT. Cell cycle was detected by flow cytometry. Cell morphology was detected by Wright's stain and transmission electron microscopy. The changes of cyclin D1, c-myc in mRNA and protein were detected by RT-PCR and western blot respectively.Results and conclusions are as follows:1. We successfully constructed pPTD-OD-HA and OD-HA prokaryotic expression vector; PTD-OD-HA and OD-HA fusion protein were successfully expressed in E.coli BL21 (DE3) stain; the optimum inducing expression conditions for PTD-OD-HA and OD-HA fusion proteins were 0.1mmol / L IPTG induced for 6h at 37℃and 1.0mmol / L IPTG induced for 4h at 37℃respectively. The purified proteins were identified by SDS-PAGE coomassie brilliant blue staining and western blot.2. PTD-OD-HA fusion protein can trigger specific apoptosis in Bcr/Abl-positive cells without affecting Bcr/Abl negative cells. Typical DNA ladders were detected. Apoptotic bodies were observed by transimmion electron microscopy. At mRNA and protein level, increasing of Bax and decreasing of bcl-2 were observed.3. Cell growth was inhibited by PTD-OD-HA protein in K562 and BaF3-P210 cells; cell cycle was arrested in G1 phase; a large number of vacuoles were observed in the cytoplasm, mitochondrial swelling, Lysosomes increased. Both cyclin D1 and c-myc decreased in mRNA and protein level.