[Construction of a site-directed mutant of R555W-BIGH3 and its influence on human corneal epithelial cells].
OBJECTIVE
To construct the eukaryotic express vector of wild type and R555W mutated transforming growth factor beta induced (BIGH3) gene, and to determine the effects of overexpression of R555W mutated BIGH3 in human corneal epithelial (HCE) cells.


METHODS
The full coding domain sequence of BIGH3 gene was cloned from human corneal tissue from operative spackman with RT-PCR. Mutagenesis of R555W-BIGH3 was performed in rapid site-directed mutagenesis technique in the expression vector pIRES2-EGFP. The two types of BIGH3 gene were transient transfected to HCE cells. Immunoblotting was used to detect the expression of BIGH3. The cell apoptosis rates were observed by flow cytometry. The morphological changes of the cells were examined by electron microscopy. The viability of caspase-3 was examined.


RESULTS
Wild type and mutated BIGH3 gene were successfully amplified by PCR. After HCE cells transfected with two types eukaryotic expression plasmid, the BIGH3 were detected in the HCE cells. The cell apoptosis were observed by flow cytometry and electron microscopy. The viability of caspase-3 was increased in the R555W mutated type.


CONCLUSIONS
The R555W mutated BIGH3 is successfully obtained by rapid site-directed mutagenesis technique. Overexpression of R555W mutated BIGH3 induces apoptosis in HCE cells through activation of caspase-3.
Ge
Hong-yan
HY
Department of Ophthalmology, First Affiliated Hospital of Harbin Medical University, Harbin 150001, China.
Song
Zhen
Z
Shi
Zhan
Z
Liu
Ping
P
D017209:Apoptosis / Q000235:genetics*; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology*; D019573:Epithelium, Corneal / Q000473:pathology; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics*; D000099222:betaIG-H3 Protein
D053148:Caspase 3; D016326:Extracellular Matrix Proteins; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein; C505407:CASP3 protein, human