| Adipose differentiation-related protein (ADFP) is a specific marker of lipid accumulation in diverse cell types and diseases. ADFP is rapidly induced early during adipocyte differentiation and absent in maturing adipocytes. ADFP specificly specifically localizes on lipid droplets, and is a member of PAT (Perilinpin, ADFP, TIP47) family, which are associated with the surface of lipid droplets. Adipophilin, homologous protein of porcine ADFP, not only participates in lipid metabolism, and stimulates lipid accumulation and lipid droplets formation, there is also a correlation between particular human diseases and accumulation of lipid droplets that suggests ADFP is a marker of pathological changes. Such diseases include atheroma, steatosis, obesity, Type 2 diabetes and some cancers. So, analysis of the properties of surface proteins has received much attention.We herein report the molecular cloning, choromosome mapping,tissue distribution and transcnptional regulation of porcine ADFP in molecular and cellular level. We cloned complete open reading frame (ORF) and non-conservative C-terminal of ADFP cDNA by Reverse transcription (RT)-PCR. The obtained 1380 bp nucleotides sequence shares 99.4% identity with the published pig ADFP, encoding a protein of 460 amino acids which shares 85.9, 76.8, 75.2, 86.8 and 82.7% identiy with human, mouse, rat, dog and cow ADFP (4 amino acids difference with the published pig ADFP), indicating ADFP is conserved during evolution and play an important physiological role. The obtained 719 bp C-terminal cDNA were digested with BamHI and Xhol and subcloned into the same sites of the bacterial expression vector pGEX-KG to produce constructs encoding the partial ADFP isoform fused in-frame with a glutathione S-transfrerase (GST) sequence. E.coli strain BL21(ED3) transformed with the constructs were used for fusion protein expression. The recombinant ADFP isoforms were expressed, purified and subjected to SDS PAGE and Western blot analyses. Then immuned New Zealand White rabbit as antigen to harvest pig anti-rabbit immunoglobulin antiserum. Intron 4, 5 and 7 were identified according to the homology of human and mouse ADFP. Genomic structure comparison of Meishan pig and Korean native pig indicates that a 276 bp SINE loss happened in intron 4. There are one more SINE in intron 7. These SINE contents, especially the lost SINE can be valuable evolution marks in diverse breeds. Using the radio hybridization panel, pig ADFP gene was located to choromosome Iq21-q27. The result is consistent with the comparative map between human and pig. Semi-quantitative PCR showed that Meishan pig ADFP mRNA was ubiquitously expressed with particularly high abundance in brain and adipose tissue, followed by testis, liver, heart, spleen, gut and muscle, and extremely low in kidney. We stimulated COS-7 cells with two concentrations of Clenbuterol Hydrochloride for 3 hours. Semi-quantitative PCR showed that in 1×10~-5 M Clenbuterol Hydrochloride, expression of ADFP in COS-7 cells was down-regulated to 30% of the control, whereas 1× 10~-4 M treatment of Clenbuterol Hydrochloride up-regulated ADFP expression to 112% of the control, suggesting the potential function of ADFP in lipid deposition. |
PDF Full Text Request