ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2009, Vol. 40 ›› Issue (7): 982-991.doi:

• 遗传繁育 • Previous Articles     Next Articles

Expression Study and Establishment of pEGFPADSL Cloning Cell Lines ofShouguang Chicken Adenylosuccinate Lyase Gene

LIU Changqing ，ZHANG Honghai ，WEN Jie 2，MA Yuehui 2* ，GUAN Weijun 2* ，TANG Xuexi 1   

  1. 1. Division of Life Sciences and Technology，Ocean University of China, Qingdao 266003,China；2. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China；3. Bioscience Department of Bengbu Medical College, Bengbu 233000, China；4. College of Life Science, Qufu Normal University, Qufu 273165, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2009-07-24 Published:2009-07-24
  • Contact: MA Yuehui，GUAN Weijun

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Abstract: The specific expression of ADSL gene in 8 different tissues of Shouguang chicken was investigated by RTPCR and RACE in this study. The fusion expression vector pGEXADSL was constructed and transformed into Escherichia coli BL21(DE3), positive cloning screened, induced and expressed by IPTG. Eukaryotic expression vector pEGFPADSL was transfected into Shouguang chicken fibroblast cells using lipofectin method. The results of sequences analysis showed that the open reading frame of 1 455 nucleotides encode a protein of 485 amino acids; The promoter of chicken ADSL cDNA showed typical features of house keeping genes，there was a C28T mutation which caused the site mutate to NRF2 binding site. The SDSPAGE electrophoresis results showed that there were specific bands, about 805 ku and an isoelectric point of 679, and Westernblot analysis showed that the fused protein was ADSL. After transfection 24, 48 and 72 h, transfection rate of pEGFPADSL were between 264%-412%, and green fluorescent could be observed in cytoplasm and nucleus welldistributed except cryptomere vesicle. Both of RTPCR and Westernblot confirmed that the pEGFPADSL had been integrated into the Shouguang chicken fibroblast cell genome, and accessed to the normal fusion protein expression. This research revealed the ADSL gene structure of excellent native chickens of China, established the foundation for further research with its biological function.