Construction,transfection and activity identification of eukaryotic expression vector of human PRKCB1 containing enhanced green fluorescence protein gene
- VernacularTitle:携带增强绿色荧光蛋白基因的人PRKCB1真核表达载体构建、转染及活性鉴定
- Author:
Lian DUAN
;
Bo ZHOU
;
Qifu LI
- Publication Type:Journal Article
- Keywords:
protein kinase C?2;
eukaryotic expression;
transfection;
green fluorescent protein;
activity
- From:
Basic & Clinical Medicine
2006;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the eukaryotic expression vector of human PRKCB1 containing enhanced green fluorescence protein gene and transfer into human umbilical vein endothelial cells(HUVECs),then identify activity of expression protein.Methods The pReceiver-M29-PRKCB1 eukaryotic expression plasmid was constructed by frame amplified from pMD18-T-PRKCB1 plasmid.Then the recombinant plasmids were identified by enzyme analysis and DNA sequencing.According to optimized conditions,the eukaryotic expression plasmids were transfered into HUVECs and observed under fluorescence microscope.After that,transfection efficiency was calculated under random vision.The plasma membrane/cytosol ratio of fluorescence was calculated under confocal microscope.The translocation was identified.Results The gene sequence was completely consistent with that reported in GenBank.The enhanced green fluorescence protein was observed in HUVECs after 48 hours.Transfection efficiency was 18.6%?1.6%.The translocation was observed.Conclusion The eukaryotic expression plasmid is successfullyconstructed and transfered into HUVECs,the translocation was identified.It is a potential tool for screening HUVECs stably expressing human protein kinase C ?2 and isolating protein complex.
