Construction Of Claudin-1 Gene Fluorescent Eukaryotic Expression Vector And Its Expression In HepG2 Hepatoma Cells

Objective Now widely recognized loss of adhesion between cells can lead to the destruction of cells connected it with tumor cell invasion and metastasis.Adhesion and tight junction connecting two connections between the epithelial cell adhesion to the main way in which the side of tight junctions at the top of cells, maintaining cells selectively permeable ions and molecules.Tight junction in the presence of a transmembrane protein 3 composed of known integration of body, including the blocking protein, adhesion molecules and Claudin protein connection.Claudin tight junction protein is the main component, and mainly through protein kinase pathway is involved in cell-cell signal transduction between. Recent studies have reported that, Claudin abnormal expression of proteins associated with the progress of malignant tumors.Claudin-1 is a tight junction protein, it belongs to the claudin family, is highly expressed in the liver and other epithelial tissues also expressed . Recent studies found, claudin-1 in the abnormal expression of tumor is closely related to the field of tumor research focus .To construct pDsRED₁-Claudin-1 plasmid, and express it in HepG2 hepatoma cells.Methods Using reverse transcription polymerase chain reaction (RT-PCR) to amplify Claudin-1 ORF gene, it was subcloned into a red fluorescent protein vector,pDsRED₁-N1. After identified by PCR,enzyme digestion and DNA sequencing,the recombinant vector pDsRED₁-Claudin-1 was then transfected into HepG2 cells. The expression of red fluorescent protein was observed by fluorescent microscope and Western blot.Results The pDsRED₁-Claudin-1 plasmid has been constructed successfully and the Claudin-1-DsRED₁ has been observed correctly.Conclusion HepG2 cells were successfully extracted total RNA, using RT-PCR technique to obtain Claudin-1 gene, use of Xho I and Bam HI digested PCR product and pDsRED₁-N1 vector to construct a recombinant plasmid, after Xho I, Bam HI digestion, protein Electrophoresis verified.A red fluorescent protein reporter gene vector containing Claudin-1 ORF gene sequence has been constructed successfully and expressed highly in HepG2 cells.