[Molecular cloning and expression of human PDGF-B chain peptide gene and its effect on cat corneal endothelial cell proliferation in vitro].
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.
Luo
Wen-juan
WJ
Department of Ophthalmology, Affiliated Hospital of Medical College, Qingdao University, Qingdao 266001, China.
Liu
Mei-guang
MG
Wang
Chuan-fu
CF
Wang
Li-mei
LM
D000818:Animals; D000077214:Becaplermin; D002415:Cats; D049109:Cell Proliferation / Q000187:drug effects*; D003001:Cloning, Molecular; D003315:Cornea / Q000166:cytology*; D004847:Epithelial Cells / Q000166:cytology*; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D010982:Platelet-Derived Growth Factor / Q000235:genetics* / Q000494:pharmacology*; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology
D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D000077214:Becaplermin