Construction and identification of luciferase reporter gene vector containing HMGB1 promoter
AIM: To construct luciferase reporter gene vectors containing high mobility group box 1 protein ( HMGB1 ) promoter and to assay the transcriptional activity of HMGB1 promoter induced by mechanical stretch in alveolar epithelial ceils (A549). METHODS: HMGB1 promoter were amplified from the genomic DNA of A549 ceils by PCR and cloned into luciferase reporter gene vector, pGl3. The reeombined vector was transfeeted into A549 cells, and the activity of the luciferase was determined after the cells were stimulated by mechanical stretch at 5% and 20% elongation. RESULTS: PCR and sequencing results indicated that the amplified sequence was correct. The results of restriction enzyme digestion indicated that the recombinant eukaryotic vector pGL3HMGB1P was successfully constructed. The iuciferase reporter system showed that the transcription activation of pGL3-HMGB1 P was 2.9 times higher than pGL3-Basic when subject to 5% stretch ( P < 0.05 ), while the transcription activation of pGL3-HMGB1 P was 6. 2 times higher than pGL3-Basic when subject to 20% stretch( P < 0.01 ). CONCLUSION: Mechanical stretch induced high transcriptional activity of HMGB1 promoter in A549 cells was confirmed by luciferase reporter gcne system, and supplied an experimental base for further study of the transcriptional regulation of HMGB1.
mechanical stretchhigh mobility group box 1 proteingenereporter
NETL
NSTL
万方数据
维普
