Study On Cloning, Expression And Biological Activities Of Chicken P15～(ink4b)

INK4a_ARF locus has a crucial role on controlling cell proliferation and tumor suppression.It can control a variety of familial and monogenesis tumors.INK4b and INK4a locate on human chromosome 9p21 in tandem(Human Genome Organization designation CDKN2B-CDKN2A).In mice and rat counterpart , they locate on chromosome 4 and chromosome 5 respectively.As an inhibitor of complex CDK4/6-cyclinD, P16 inhibits phosphrating PRb by CDK4/6-cyclinD in late G1.Thus,cell can not bypass G1/S restriction point.Accumulating research evidence indicates that INK4a/4b evolves from gene repetition.Gene INK4a evolves from gene INK4b.Thus, gene INK4b is more primitive than gene INK4a .Soo-Hyun and his parterners have isolated and characterized the equivalent locus in chickens in 2002.Surprisingly, they found that orthologues of INK4b and ARF, chickens do not encode an equivalent of INK4a.From above discussion,we can make hypothesis that P15 have a strong role in non-P16 chicken cells.As follows,this research was divided two parts.Cloning and expression chicken p15^ink4b geneThe chicken p15^ink4b gene was amplified by PCR, then the PCR product and vector pVAX1 digest with double enzyme BamHI and XhoI, chicken p15^ink4b gene was cloned into a eukaryotic expression vector pVAX1.Then, we identifided positive clone by PCR and bienzymatic digestion and furtherly identified by sequencing.Sequencing result was analyzed by biological software. It was confirmed that the gene sequence was consistent with the sequence in GENEBANK.Positive recombinant eukaryotic plasmids were transfected into CEFs. We detected transcription and expression of p15^ink4b by employed RT-PCR and westernblot in CEFs.Chicken p15^ink4b gene inhibited proliferation of CEFs, arrested CEFs in G1/G0 and induced cellular senescence of CEFsWe transfected pVAX1-p15 into CEFs by Lipofectamine2000. After transient transfection,proliferation of CEFs was determinated by direct cell number counting and MTT assay at 24, 48, 72 hours respectively. Cell cycle arrest was detected by fluorescence activated cell sorter (FACS) analysis.Cellular senescence was determinated by detecting SA-β- galactosidase. Results show that P15 can express in CEFs, nhibit cell cycle progression of CEFs at G1/S restriction point and induce cellular senescence CEFs.In conclusion,this research explored biological activities of P15 in CEFs initially, which would lay a foundation to the researches in mechanism of cell senesence,cell cycle regulation and tumor genesis.