Abstract: To detect the over-expression of LPAR1 and the accumulation of cAMP in transfected 293T cells, human LPAR1 gene was cloned and the LPAR1 expressing vector was constructed. LPAR1 CDS sequence was cloned from human gastric cancer cells and sub-cloned into the pCR2.1 vector. By sequencing, digestion and ligation, LPAR1 was directionally cloned into eukaryotic expression plasmid pIRES2-EGFP. The reconstructed plasmid was identified with enzyme digestion and sequencing；the plasmid was transfected into 293T cells with lipofectamine2000. Real-time PCR was performed to assess exogenous LPAR1 expression in 293T cells. ELISA was used to detect the activity of LPAR1. These results indicate that the expression vector containing human LPAR1 gene has been established and LPAR1 gene has also been over-expressed successfully in 293T cells. Through the signaling pathways downstream of LPAR1 mediated under the stimulation of LPA demonstrates the activity of the cloned gene expression of LPAR1 protein. The establishment of LPAR1-overexpressing cell model, not only provides the foundation to research the function of LPAR1 in the next step, but also make it possibly for quantitative the concentration of LPA through the linear relationship between LPA and cAMP.

Key words: LPAR1, Gene cloning, Transgenic cell model, cAMP response