| Popis: |
The full-length of the cDNA sequence of Beijing fatty chicken ADSL gene was investigated by RT-PCR and RACE in this study. The results demonstrated that the complete ADSL cDNA revealed an open reading frame of 1455 nucleotides coding for 485 amino acid residues. The promoter of chicken ADSL cDNA showed typical features of house-keeping genes: no canonical TATA and CAAT boxes, 72.65% GC in 234 bp near the start codon, and there was a C-28T mutation, which caused the site CTCC mutating to the NRF-2 binding site CTTC. The complete open reading frame of the ADSL gene was inserted into expression vector pGEX-4T-1 and the fusion expression vector pGEX-ADSL was constructed. The pGEX-ADSL was transformed into Escherichia coli BL21 (DE3), positive cloning screened, induced and expresses by IPTG. The SDS-PAGE electrophoresis results showed that there were specific bands, about 80.5 kD and an isoelectric point of 6.79, which reached 26.9% of total cell proteins induced in 5hrs, and was an insoluble inclusion body. Under optimal conditions, ADSL was purified by Glutathione Sepharase 4B affinity chromatography, and Western blotting analysis showed that the fused protein was ADSL. This research was a foundation for further research into ADSL’s biological function and its application identification. |
