Cloning Of The Partial Important Traits Gene Of Beijing Fatty Chicken And Construction Of 3 Kinds Of PURH Expression Vector

PURH, CD3δ, CD3ε, CD8α, IFNαreceptor1, IFNαreceptor2, IFNγreceptor2, RBMX, PEPD, WRB, the genes of Beijing fatty chicken,were obtained by RT-PCR amplification.Using the organization to adherent culture,we succeeded in constructing Beijing fatty chicken fibroblast specific target cell.The specific expression of PURH gene in 8 different tissues of Beijing fatty chicken was investigated by RT-PCR in this study. The full length of PURH cDNA was inserted into fusion expression vector pEGFP-N3,pEYFP-N1 and pDsRed1-N1 multiple cloning sites between EcoR I and BamH I, and construct recombinant eukaryotic expression vector pEGFP-N3- PURH,pEYFP-N1- PURH and pDsRed1-N1- PURH with GFP as reporter gene. We used lipofectin method to transfect the recombinant vectors into Beijing fatty chicken fibroblast cells. After the G418 screening and resistant colonies was picked and subcultured until use. The results showed:1. Ten genes were sequenced and submitted to Genbank.2. According to ATCC cell storehouse appraisal content and evaluation criteria, the result indicated that. The cell heredity performance is stable, indicated had not had the species crossing pollution and the malignant transformation.3. 24, 48 and 72h after transferring, the expression efficiency of 3 kinds of recombinant fusion protein genes were between 10.3%-53.2%, and the fluorescence could be observed in cytoplasm and nucleus well-distributed except cryptomere vesicle; Through the G418 drug screening and monoclonal training, three cell lines of stable expression of pEGFP-N3-PURH,pEYFP-N1-PURH and pDsRed1-N1-PURH fusion protein was cloned. RT-PCR and western blot both confirmed the pEGFP-N3-PURH, pEYFP-N1-PURH and pDsRed1-N1-PURH have been integrated into the Beijing chicken fibroblast cell genome.