[Construction of the eukaryotic expression vector pEGFP-claudin-1 and its expression in 293T cells].
OBJECTIVE
To construct the eukaryotic expression vector pEGFP-claudin-1 and to make it express expressed in 293T cells.


METHODS
Claudin-1 ORF was amplified by reverse transcription polymerase chain reaction (RT-PCR). The eukaryotic expression vector pEGFP-claudin-1 was constructed by introducing claudin-1 DNA fragment into the sites of Xho I and BamH I of pEGFP-C3 vector. The plasmid was transfected into 293T cells with lipofectamine. The expressed EGFP was observed by fluorescent microscope. The EGFP-claudin-1 was detected by Western blot.


RESULTS
The eukaryotic expression vector pEGFP-claudin-1 was constructed successfully. The expression of EGFP-claudin-1 was observed in the membrane of transfected cells by fluorescent microscope and a 49 kD protein was detected by Western blot.


CONCLUSIONS
The recombinant expression vector pEGFP-claudin-1 has been successfully constructed and claudin-1 gene can be expressed in 293T cells. This study lays a foundation for further research into the function of claudin-1.
Wei
Xin
X
Department of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China. weixinxp@gmail.com
Zhang
Ye
Y
Li
Jun
J
Ma
Li
L
Pan
Lei
L
Wang
Ping-zhong
PZ
Bai
Xue-fan
XF
Jia
Zhan-sheng
ZS
D000818:Animals; D015153:Blotting, Western; D062445:Claudin-1; D003001:Cloning, Molecular; D006224:Cricetinae; D056890:Eukaryota / Q000235:genetics* / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D008055:Lipids / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods; D019444:Vaccines, DNA
C568108:CLDN1 protein, human; D062445:Claudin-1; D008055:Lipids; C086724:Lipofectamine; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D019444:Vaccines, DNA