The Molecule Mechanism Research Of Biphenyl Hydrolase-Related Protein（BPHL） In The Development Of APl

PART ⅠIdentification of interaction between BPHL and PML-CObjective To explore the interaction between BPHL and PML-C byco-immunoprecipitation and yeast two-hybrid.Methods The recombination expression plasmids pGBKT7-PML-Cand pACT2-BPHL were co-transformed into yeast AH109, to investigatethe interaction in vivo. the expression vector of HA-tagged fusion protein(PCMV-HA-PML-C) and the expression vector of Myc-tagged fusionprotein (PCMV-myc-BPHL) were respectively constructed, identified andtransfected into human embryo kidney293(HEK293) cells. the interactionbetween PML-C and BPHL was investigated by co-immunoprecipitationin vitriol.Results Blue clones were found in QDO/X-α-gal plate,eukaryoticexpression vectors of PCMV-HA-PML-C and PCMV-myc-BPHL weresuccessfully constructed and showed correctly by double restrictionenzyme digestion.They were co-transfected into HEK293cells.ThenHA-PML-C was immunoprecipitated by anti-HA polyclonal antibody,Myc-BPHL was identified by western blotting with anti-Myc monoclonal antibody from immunoprecipitated complex.Conclusion The eukaryotic expression vector of PCMV-HA-PML-Cand PCMV-myc-BPHL were constructed successfully.The interactionbetween PML-C and BPHL was identified by co-immunoprecipitation andyeast two-hybrid. PARTⅡIdentification of interaction between BPHL and PML(totallength of no NLS of PML)Objective To explore the interaction between BPHL and PML byco-immunoprecipitation.Methods The expression vector of HA-tagged fusion protein(PCMV-HA-PML) and the expression vector of Myc-tagged fusionprotein (PCMV-myc-BPHL) were respectively constructed, identified andtransfected into human embryo kidney293(HEK293) cells. Theinteraction between PML and BPHL was investigated byCo-immunoprecipitation in vitriol.Results eukaryotic expression vectors of PCMV-HA-PML andPCMV-myc-BPHL were successfully constructed and showed correctly byDouble restriction enzyme digestion.They were co-transfected into HEK293cells.Then HA-PML was immunoprecipitated by anti-HA polyclonal antibody, myc-BPHL was identified by western blot with anti-Mycmonoclonal antibody from immunoprecipitated complex.Conclusion The eukaryotic expression vector of PCMV-HA-PMLand PCMV-myc-BPHL were constructed successfully.The interactionbetween PML and BPHL was identified by co-immunoprecipitation. PART ШConstruction of BPHL si-RNA expression vector and itsexpression in NB4cellObjective To construct the recombinant vector si-RNA of BPHLgene and to explore its expression in the NB4cell.Methods According to the encoding sequence of BPHL, the si-RNAoligonucleotide sequence was designed,synthesized and cloned into theplasmid vector pGPU6. the recombinant vector pGPU6-BPHL si-RNAwas transfected into NB4cells. The expression of BPHL mRNA andprotein level were detected by RT-PCR and Western blotting after boltingby G418.Results The recombinant vector pGPU6-BPHL si-RNA gene wassuccessfully constructed. The expression of transcription and translationlevels of BPHL gene in NB4cells were significantly declined.Conclusions The recombinant vector pGPU6-BPHL si RNA significantly inhibited the transcription and translation levels of BPHLgene. PART ⅣEffect of silencing BPHL on the proliferation and apoptosis ofNB4cell by RNA interferenceObjective To explore the effect of silencing BPHL gene on theproliferation and apoptosis of NB4cell by RNA interference.Methods The recombinant vector pGPU6-BPHL-1.1si-RNA,negative vector pGPU6-N.1and the empty vector pGPU6were transfectedinto NB4cells. The cell growth was measured with MTT essay and thecell cycle and apoptosis was examined by flow cytometry.Results TO compare with the negative group and blank group, thegrowth of NB4cell of the experimental group decrease,the percentage ofG0/G1stage cells were significant higher, while the percentage of S stagecells was significant lower. The apoptosis rate of experimental cell wassignificantly higher than that of control group (P<0.01)Conclusion Down-regulation of BPHL can inhibit the growth ofNB4cell, change the cycle of it and induce the cell apoptosis.