摘要
Cyclin A is a key regulator of the cell cycle. Its expression may become disrupted in virusinfected cells, leading to deregulation of the cell cycle and increased cell proliferation. Here, we cloned the porcine cyclin A gene and verified its functionality in swine umbilicus vein endothelial cells （SUVEC）. The human cyclin A gene was used to probe databases to clone the pig cyclin A gene electronically. The identified porcine cDNA contained an open reading frame of 1,299 bp, encoding 432 amino acids, the same length as the human cyclin A protein. The porcine cyclin A gene comprises eight exons on chromosome 8. The sequence of the in silico clone and expression of this novel gene were confirmed in SUVEC by reverse transcription PCR. Western blotting of cell lysates from SUVEC transfected with a cyclin A enhanced green fluorescent protein （EGFP） fusion construct revealed a band at approximately 40 kDa. Confocal microscopy of CycA-EGFP-expressing cells showed that the fusion protein was expressed in the nucleus. Flow cytometry demonstrated that more stably expressing SUVEC-CycA-EGFP were in G1 phase （15% to 20% increase） and fewer were in S phase （18% decrease） compared with control ceils. MTS assays showed that the proliferative activity of SUVEC-Cy- cAG-EGFP was significantly higher than that of the control cells. In conclusion, we have cloned the pig cyclin A gene and demonstrated that its biological function is consistent with cyclin A in other mammali- an species. This provides a foundation for future research on the impact of virus infection on cyclin A.
Cyclin A is a key regulator of the cell cycle. Its expression may become disrupted in virusinfected cells, leading to deregulation of the cell cycle and increased cell proliferation. Here, we cloned the porcine cyclin A gene and verified its functionality in swine umbilicus vein endothelial cells （SUVEC）. The human cyclin A gene was used to probe databases to clone the pig cyclin A gene electronically. The identified porcine cDNA contained an open reading frame of 1,299 bp, encoding 432 amino acids, the same length as the human cyclin A protein. The porcine cyclin A gene comprises eight exons on chromosome 8. The sequence of the in silico clone and expression of this novel gene were confirmed in SUVEC by reverse transcription PCR. Western blotting of cell lysates from SUVEC transfected with a cyclin A enhanced green fluorescent protein （EGFP） fusion construct revealed a band at approximately 40 kDa. Confocal microscopy of CycA-EGFP-expressing cells showed that the fusion protein was expressed in the nucleus. Flow cytometry demonstrated that more stably expressing SUVEC-CycA-EGFP were in G1 phase （15% to 20% increase） and fewer were in S phase （18% decrease） compared with control ceils. MTS assays showed that the proliferative activity of SUVEC-Cy- cAG-EGFP was significantly higher than that of the control cells. In conclusion, we have cloned the pig cyclin A gene and demonstrated that its biological function is consistent with cyclin A in other mammali- an species. This provides a foundation for future research on the impact of virus infection on cyclin A.
