Abstract:

BACKGROUND: Claudin-1 is a multi-functional protein; besides, construction of tight junction strand to seal paracellular space, which expression disorder in transcriptional level may be also involved in malignant cancer for invasion, metastasis and prognosis as a molecular marker.
OBJECTIVE: To construct recombinant rat Claudin-1 luciferase reporter plasmid.
METHODS: Oligonucleotide containing about 2 000 bp in 5’-UTR of rat Claudin-1 genome DNA was designed and synthesized, which was inserted into pGL3-Basic vector after double digestion by restriction enzyme KpnⅠ and MluⅠ; competent E.coli DH5α and pMD18-T-simple vector were used for screening of positive sample. Positive clones were identified by PCR and sequencing. There were four groups in the experiment: Control group, positive control group (transfected by pGL3-promoter plasmid), negative group (transfected by pGL3-Basic plasmid) and experimental group (transfected by pGL3-Claudin-1 promoter plasmid). Claudin-1 promoter activity was detected in the 293T cell transiently transfected.
RESULTS AND CONCLUSION: The result of recombinant pGL3 plasmid from DNA sequencing was fully consistent with promoter sequence of rat Claudin-1 gene in GenBank (gi|62750811) by NCBI blast assay. Compared with pGL3-basic plasmid, the transcriptional activity of recombinant luciferase report gene in recombinant pGL3 plasmid was obviously increased (P < 0.001). Gene sequencing and transfection results confirmed that effective pGL3-Claudin-1 promoter plasmid had been constructed successfully.