Construction of Vectors Expressing Inhibiting Peptides for Nuclear Import and Its Effect on Growth and Migration of HeLa Cell
Abstract
To construct the expression vectors for red fluorescent protein fused with inhibiting peptides for nuclear import (Bimax), and explore the location of Bimax and its potential effects on cell proliferation and migration in HeLa cells. Methods Two kinds of polynucleotide encoding inhibiting peptides for nuclear import were synthesis respectively and subsequently annealed for inserting into vector pDs-Red-C1. The recombinant plasmids were transfected into competent bacterial DH-5α. After transfection, the positive bacteria were picked up for DNA sequencing. The recombinant plasmids pDs-Red-Bimax2, pDs-Red-Bimax1 and negative plasmid pDs-Red-C1 were transfected into HeLa cells respectively according to Lipofectamine2000 protocol. After transfection, the expression and location of red fluorescent protein were observed with fluorescence microscope. Furthermore, MTT assay and cell-migration assay were used to detect the proliferation and migration of Bimax transducted cells. ResultsDNA sequencing showed that the polynucleotides encoding Bimax1 or Bimax2 were inserted into pDs-Red-C1 vector successfully. After transfected into HeLa cells, the inhibiting peptide induced red fluorescent protein locating in nuclear. Furthermore, either the fusion protein RFP-Bimax1 or RFP-Bimax2 can suppress the proliferation and migration of HeLa cells. Conclusion The expression vectors for red fluorescent protein fused with inhibiting peptides for nuclear import were successfully constructed. In addition, the fusion proteins were expressed and located in nuclear and suppressed the proliferation and migration of tumor cells.
Keywords: Nuclear import, Red fluorescent protein,ProliferationMigration
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