Cloning Of Porcine CDK9 Gene And Its Preliminary Functional Investigation

Cyclin-dependent kinases play a key regulatory role in the orderly process of cell cycle. Thirteen members of Cyclin-dependent kinases(CDKs) have been identified so far. CDK9 is a cdc2-like Ser/Thr kinase whose complexes with T and K type cyclins(T1,T2a,T2b,K) constitute the basal transcription elongation factor b(P-TEFb), which promotes transcriptional elongation by phorsphorylating the carboxy-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymeraseⅡand other positive transcription elongation factor (NELF and N-TEFs). Investigation proves that CDK9 is a core transcriptional factor in the process of elongation. Therefore, it is readily apparent that CDK9 plays important roles in the regulation of a variety of physiological processes, especially in the immune responses, inflammation, cell growth and differentiation and viral replication. In quest of the CDK functions in pig, we cloned porcine CDK9 gene in both electronical and molecular methods, and made a preliminary study on CDK9 expression pattern in swine umbilical vein endothelial cells (SUVEC) and E. Coli. Our results are the followings.1. Based on human CDK9 gene sequence as a homologous reference, we in silico cloned porcine CDK9 gene and experimentally confirmed it with accession number of GQ449385. Our results suggested that the full length of porcine CDK9 is 1 820 bp with ORF of 1 119 bp. Porcine CDK9 encodes 372 amino acids and locates on the chromosome 1.2. CDK9 fragment was inserted into eukaryotic expression vector pEGFP-C1 which contains a green fluorescence reporter gene, thus construting a recombinant plasmid pEGFP-CDK9. The recombinant plasmid and the control plasmid pEGFP-C1 were transfected to SUVECs by Lipofectamine 2000 Regent. Fourty eight hours post transfection, our targeted gene was successfully expressed in SUVECs, and then confirmed by fluorescence microscope and western blot. Compared with the control group, experimental group cells showed no significant change during the whole stage of cell cycle.3. We subcloned porcine CDK9 gene into an expression vector pET-32a(+), thus constructing the recombinant expression plasmid pET32a-CDK9. Then it was transformed into E.coli BL21 with CaCl2 method, and induced by 0.1mmol/L IPTG at 28℃for 7 hours. The recombinant CDK9 was expressed efficiently, which was confirmed by SDS-PAGE assay. The fusion protein had a molecular weight of approximate 62ku. Recombinant CDK9 protein mainly expressed in form of inclusion bodies.These results provide novel rationale and references for further investigation on CDK9 gene regarding its in vitro expression and biological activity including the regulatory role in cell cycle.