[Cloning of tight junction protein claudin-1 and construction of the mammalian expression vector].
OBJECTIVE
To construct a recombinant plasmid containing the coding region of tight junction protein claudin-1 gene to understand the functional role of claudin-1 in human colorectal carcinoma.


METHODS
The total RNA was extracted using Trizol from human colorectal carcinoma cell line SW620, and the DNA for claudin-1 was obtained by means of RT-PCR. The PCR product was inserted into the plasmid pEGFP-C1 after restriction endonuclease digestion and ligation. The recombinant plasmid was then transfected into human colorectal carcinoma cell line SW480.


RESULTS
The sequence of the recombinant plasmid was verified by restriction endonuclease and DNA sequence analysis, and the target protein expression was detected mostly on the cell membrane.


CONCLUSIONS
The expression vector claudin-1/pEGFP-C1 has been constructed successfully and the target protein can be expressed in human colorectal carcinoma cell line.
Chen
Lin
L
Institute for Digestive Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. clin66666@yahoo.com.cn
Jiang
Bo
B
Zhang
Ya-li
YL
Zhang
Hong-quan
HQ
Gong
Wei
W
Lu
Qing
Q
D045744:Cell Line, Tumor; D062445:Claudin-1; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions / Q000378:metabolism*; D014162:Transfection
C568108:CLDN1 protein, human; D062445:Claudin-1; D008565:Membrane Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins