Construction of recombinant lentiviral vectors containing Rheb gene and its mutant Rheb'D60K gene and their expression in human liver cancer cells.
- Author:
Ke-He CHEN
1
;
Bo LIANG
;
Zhen-Hong ZOU
;
Ze-Long HAN
;
Jin-Fei PAN
;
An-Ling LIU
Author Information
- Publication Type:Journal Article
- MeSH: Apoptosis; genetics; Carcinoma, Hepatocellular; metabolism; pathology; Genetic Vectors; genetics; HEK293 Cells; Humans; Lentivirus; genetics; metabolism; Liver Neoplasms; metabolism; pathology; MCF-7 Cells; Monomeric GTP-Binding Proteins; biosynthesis; genetics; Mutant Proteins; genetics; Neuropeptides; biosynthesis; genetics; Ras Homolog Enriched in Brain Protein; Recombinant Proteins; biosynthesis; genetics; Transfection
- From: Journal of Southern Medical University 2012;32(3):341-344
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct recombinant lentiviral vectors carrying Rheb gene and its mutant Rheb'D60K gene, and examine their expression in human liver cancer cells.
METHODSRheb gene was amplified by PCR to construct the recombinant plasmid LV31-Rheb-WT and LV31-Rheb-D60K. HEK-293 FT cells were contransfected with the recombinant lentiviral vector together with a lentiviral package plasmid to produce the lentiviral particles. The expression of PS6 protein was detected in the lentivirus-infected MCF-7 cells. The apoptosis of SK-HEP-1 cells transfected with LV31-Rheb-WT or LV31-Rheb-D60K was observed.
RESULTSThe recombinant LV31-Rheb-WT and LV31-Rheb-D60K vectors were confirmed by PCR and DNA sequencing. Western blotting showed that PS6 protein expression was increased in LV31-Rheb-WT-transfected cells while decreased in LV31-Rheb-D60K-transfected cells. LV31-Rheb-D60K-transfected SK-HEP-1 cells showed more obvious apoptosis after starvation than LV31-Rheb-WT-transfected cells.
CONCLUSIONLentiviral vectors carrying Rheb gene and its mutant has been successfully constructed, which can be useful in further investigation of the role of Rheb gene in cancer cells.
