A Method Of Relaxin2 Activity Assay Based On Stably Transfected RXFP1 Cell Line

Relaxin2 is a peptide hormone that belongs to the insulin family and consists of 53 amino acids condensed into two short peptide chains.Relaxin2 binds to the relaxin family peptide receptor 1(RXFP1)and exerts its biological effects in cells mainly through the cyclic adenosine monophosphate(c AMP)pathway.In this study,we established an ELISA assay for analyzing the biological activity of Relaxin2 based on a stably transfected RXFP1 cell line HEK293/RXFP1.The recombinant plasmid p LJM1-EGFP-RXFP1 was constructed using lentivirus plasmid p LJM1-EGFP as a vector.HEK293 T cells were used as packing cells and liposome transfection method was used.ps PAX2 and p MD2.G lentivirus were used as packing plasmids and co-transfected with recombinant plasmid.The lentivirus containing the recombinant plasmid was collected,concentrated and infected with HEK293 cells.The monoclonal recombinant engineered cell line HEK293/RXFP1 was screened by using G418 antibiotics with limited dilution method.The expression of EGFP on the cell membrane of the recombinant engineered cell line HEK293/RXFP1 was observed by fluorescence microscopy,and the expression of RXFP1 in the recombinant engineered cell line HEK293/RXFP1 was detected by Western Blot.By ELISA assay,different concentrations of Relaxin2(3.2 pg/m L?50 ng/m L)can induce different contents of c AMP in recombinant engineered cell line HEK293/RXFP1.The higher the concentration of Relaxin2 was,the more c AMP was produced.The biological activity of Relaxin2 was determined by comparing with the c AMP standard curve.This study provides a new approach for Relaxin2 activity assay,which has the advantages of sensitivity,high efficiency,high operability,and low cost.