Cloning And Prokarvotic Expression Of Thyroid Hormone Responsive Gene (SPOT14) Of Porcine

Thyroid hormone responsive SPOT14gene, which is a nuclear geneinduced by thyroid hormone, expressed predominately in adipose tissue and liver. It isinvolved in transcriptional regulation of rate-limiting enzymes in lipogenic pathway.It plays an an important role on the regulation of animal lipogenesis. In order tofurther study the function of the porcine SPOT14, the primer was designed based onthe in silico sequences information. The porcine SPOT14gene was cloned by RT-PCR,and its tissue distribution was detected. The recombinant vector pET-28α (+)-S14wasconstructed and fusion was induced expressed successfully. The protein wasidentified by Western blot. The molecular weight is20.91kD. The results show that:1. The cloning, sequence analysis and tissue distribution of the porcine SPOT14The cloned SPOT14gene contains a complete open reading frame (ORF), and itssequence length is453bp, and the coding protein include150amino acid residues,which contains35acidic amino acids and23basic amino acids, the molecular weightis17.08kD and the isoelectric point is6.15. Identity analysis shows that the SPOT14predicted peptide sequence in sus scrofa shared76.9%,80.8%,76.2%,76.2%,82.1%,32.0%homology with that of Homo sapiens, Bos taurus, Rattus norvegicus, Musmusculus, Oryctolagus cuniculus, Gallus gallus respectively. The phylogenetic treedemonstrates that sus scrofa is close to cattle. Semi-quantitative RT-PCR analysisshowed that the expression level of SPOT14is the highest in liver and adipose tissue,the lower in cerebrum, spleen, stomach, skin, caecum and rectum, the lowest in lung,duodena jejunum and ileum,without in Kidney and muscle.2. The construction and identification of the porcine pET-28α（+）-S14The primer was designed based on the clone sequence in order to expresssuccessfully S14in E.coli.. The ORF of the porcine SPOT14was subcloned intopET-28a (+) vector and this recombinant vector was identified by the bacteria PCR,double digestion and sequencing. The results show that the location, size, and correctreading frame of the porcine SPOT14are all right. Then the prokaryotic expressionvector was constructed successfully, named pET-28α (+)-S14.3. The expression of the porcine SPOT14in E.coliThe recombinant vector pET-28α (+)-S14was transformed into BL21E.coli. to determine the best conditions of protein expression, it was induced by differentconcentrations of IPTG and time.The induced protein was collected and the molecularweight of the target protein is21kD by SDS-PAGE. The fusion protein was mainlyexpressed in the inclusion body.4. The purification and identification of the porcine pET-28α（+）-S14The obtained inclusion bodies was purified by6xHis Ni-NTA affinitychromatography and a single target band was identified by SDS-PAGE. Western blotshowed that the molecular weight of the target protein(20.91kD) is21kD. Theprimary antibody was mouse anti-HisTag monoclonal antibody and the secondantibody was goat anti-mouse lgG-HRP.