Objective: To construct pET-PDGF-B plasmid with over expression of PDGF and investigate its effects on cat corneal endothelial cells proliferation in vitro.
Methods: Gene rearrangement technique was used to construct the prokaryotic vector with insertion of pET-PDGF-B. The recombinant PDGF-B was obtained from E. coli BL21 (DE3). The effects of recombinant PDGF-B on proliferation and morphologic changes of cultured cat corneal endothelial cells were assayed by modified tertrozalium salt (MTT), inverted phase contrast microscope and transmission electron microscope.
Results: PDGF-B peptide gene was inserted into prokaryotic expression vector pET-28a (+), which was confirmed by sequence analysis; a 27,000 protein band was demonstrated as pET-PDGF-B from extraction of E. coli BL21 (DE3) using western blot; MTT assay showed PDGF-BB promoted the proliferation of cat corneal endothelial cells.
Conclusions: A constructed pET-PDGF-B plasmid vector over expression of PDGF is produced. Recombinant PDGF-BB promotes cultured cat corneal endothelial cells proliferation.