Abstract:

Objective To construct the glucose-regulated protein 78 (GRP78) over-expressed plasmid, transfect human umbilical vein endothelial cells (HUVECs) in vitro, and observe its expression and location. Methods Primers were designed according to the gene information on GRP78 in PubMed, and the GRP78 gene was amplified by Polymerase Chain Reaction (PCR). By using the gene recombination technology, the double enzyme digested GRP78 gene was cloned into the pLVX-IRES-ZsGreen1 vector with GFP and the CMV-10 vector with Flag tag. Recombined plasmids were identified with enzyme digestion and sequencing and were transfected into HUVECs. Immunofluorescence assay was used to detect its expression and location. Western blotting was used to confirm its over-expression. Results The results of enzyme digestion and sequencing revealed that the recombinant plasmids pLVX-GRP78-IRES-ZsGreen1 and CMV-Flag-GRP78 were successfully constructed. After transfecting HUVECs with the successfully constructed plasmid pLVX-GRP78, a large amount of green fluorescence was observed. After transfecting HUVECs with plasmid Flag-GRP78, immunofluorescence assay revealed that Flag-GRP78 protein located in endoplasmic reticulum. Western blotting showed the successful over-expression of pLVX-GRP78 and Flag-GRP78. Conclusion The recombinant plasmids pLVX-GRP78-IRES-Green1 and CMV-Flag-GRP78 were successfully constructed and were transfected into HUVECs. Flag-GRP78 protein was observed to locate in endoplasmic reticulum.

Key words: glucose-regulated protein 78, plasmid construction, human umbilical vein endothelial cell,
endoplasmic reticulum