Study On Cloning, Expression And Effect Of Chicken P15～(INK4B) Gene

To research the chicken cell cycle regulation mechanism ,studies oncloning ,expression and effect of p15 gene were carried out in this study. Inmammalian cell both p15 and p16 are two important INK4-family inhibitors, namedfor their ability to inhibit CDK4 and induce growth arrest by specifically targeting andinactivating CDK4 and CDK6 .Recently,it has been found that p15 but not p16 ispresent in chicken cells .It is indicated that p15 could play more important roles in thecontrol on chicken cell proliferation and in tumor suppression.The length of chicken p15 gene,which is spliced by two exon transcription, is420bp, the molecular weight of its protein product is 14.5kDa. The full length p15gene was ligated by overlap PCR with five couple primers designed in accordancewith the sequence of p15 gene in GenBank.The recombinant prokaryotic expressionvector of p15 gene was constructed and expressed p15 fusion in Rosseta(DE3) E.coli .The purified p15 fusion protein as antigen immunized BALB/c mice. Hybridoma(1C6 and 3F5) stably secreting monoclonal antibody(McAb) against p15 protein wasproduced by fusion of SP2/0 myeloma cells and spleen cells of syngeneic mice whichhad been previously immunized by p15 protein. Their titers in cell culture supernatantwere 1:820 and 1:680 respectively, titers in ascites were 1:4×106 and 1:7×105respectively, and affinity constants were 3.13×109 and 1.24×108 L/mol respectively.The monoclonal antibodies were of high affinity and different antigen binding epitope,and they could specifically bind to p15 protein purified from prokaryotic expressingproduct and expressed in BAC-TO-BAC Baculovirus Expression Systems but notother cell proteins.To study the biological function of p15, recombinant eukaryon expression vector,pcDNA3.1(+)-p15,was constructed and transfected into MDCC-MSB1,a chickenlymphoblastoid cells line transformed by Marek's disease virus .G418 was used toselect the cells transfected with pcDNA3.1(+)-p15 and pcDNA3.1(+) respectively.Thecells were collected to measure the proliferation ability ,population doubling time , cellcycle phases, telomerase activity and p15 protein .In contrast to the cells transfectedwith pcDNA3.1(+) ,the cells transfected with pcDNA3.1(+)-p15 stably expressed p15protein even until 144 hours after transfection.The p15 protein induce MDCC-MSB1cell proliferation inhibition, and inhibition ratio is from 45 to 74 percent.Thepopulation doubling time of cells transfected with pcDNA3.1(+)-p15 are delay from27 hours to 416 hours. Flow Cytometry was used to distinguish cell cycle phases andit was found that p15 cause cell cycle arrest in G0/G1 phase , decrease the ratio ofcells in S and G2/M phase and strongly inhibit telomerase activity , which could not bedetected by TRAP-silver assay although some cells in S and G2/M phase.To characterize the properties and functions of p15 protein , BAC-TO-BACBaculovirus Expression Systems was used to express active p15 protein. The multipleclone site of donor plasmid in this system was reconstructed. The recombinant p15donor plasmids fused TAP-tag for purification are constructed ,pFAST-NMCS-N-TAP-p15 and pFAST-NMCS-C-TAP-p15. DH10Bac competentcells, which contain the bacmid, were transformed by two recombinant donor plasmidrespectively. The recombinant Bacmid-N-TAP-p15 and Bacmid-C-TAP-p15transformants are isolated after three times monoclonal plate. Two recombinantbacmid are transfected into sf9 cells . The recombinant virus was harvested from cellculture medium at 72 hours after transfection.The titer assay was performed afteramplifying the recombinant baculoviral stock.. sf9 cells are infected the high titerrecombinant baculovirus. The result show that the titers of N-TAP-p15 andC-TAP-p15 recombinant baculovirus is 3.6×107 pfu/mL and 5.4×107 pfu/mLrespectively. Two p15 fusion proteins were expressed in sf9 cells and amounting to4.32% and 4.7 respectively. Western Blot show that both of them can specifically bindto p15 McAb and rabbit IgG.In a word, all above data indicated that chicken p15 induce MDCC-MSB1 cellcycle arrest and inhibit the telomerase activity.Two subclass McAb against p15 proteinwere preparated. p15 fusion protein was stably expressed in BAC-TO-BACBaculovirus Expression Systems.All of these results would promote farther researcheson p15 factor and chicken cell cycle regulation mechanism.