Generation And Identification Of PD-1 SsDNA Aptamers Based On CELL-SELEX Technique

Objective: To construct a cell line with stable expression of human programmed death-1(PD-1)extracellular domain protein,and to select an aptamer with highly affinity and specificity for PD-1 extracellular domain protein by Cell-SELEX.Method: The recombinant plasmid containing human PD-1 extracellular domain(PD-1N)sequence was constructed by molecular cloning technology,and transfected into L02 cells.L02-PD-1N cells,with stable expression of PD-1 extracellular domain protein,were selected by G418,and confirmed by Western blotting and flow cytometry.An ss DNA library was constructed by chemical synthesis.The center sequence of the library was a random sequence of 30 bases,with fixed primer sequences at both ends.By Cell-SELEX technique,PD-1N specific aptamers were selected.L02-PD-1N cells,with highly expression of PD-1 extracellular domain protein,were used for positive selection.L02 cells were used for negative selection.After 12 rounds of screening,PCR amplification,plasmid transfection,monoclonal selection and sequencing were carried out to obtain candidate aptamers.The affinity of the selected aptamers for PD-1N protein and L02-PD-1N cell was verified by ELONA and flow cytometry respectively,with chemically synthesized aptamers 5’labeled with biotin or FITC.The aptamer with high affinity was selected and Kd value was determined by flow cytometry.The aptamer with the highest affinity for L02-PD-1N cells was identified,and cell immunofluorescence technique was used to evaluate its binding specificity.Subsequently,we used tissue immunofluorescence technique to detect the binding of the aptamer to PD-1 on the surface of T cells in tuberculosis and tumor patients.Result: The recombinant plasmid containing PD-1 extracellular domain gene was constructed and successfully transfected into L02 cells.L02-PD-1N cells with stable expression of PD-1 extracellular domain protein was successfully selected by G418.After 12 rounds of SELEX screening,26 valid aptamer sequences were successfully obtained.The specificity and affinity of P13 aptamer was the highest as determined by ELONA and flow cytometry,with Kd value of 89.5±24.4 n M,which was in nmol level.It was found that P13 aptamer could specifically bind to the surface of L02-PD-1N cell membrane,but not to LO2 cells,by immunofluorescence technology.Tissue immunofluorescence experiments showed that aptamer P13 can bind to PD-1molecules on the surface of T cells in tuberculosis and tumor tissues.Conclusion: We have successfully prepared L02-PD-1N cells expressing PD-1extracellular domain protein,the aptamers specific for PD-1N were screened by cell-SELEX technology using the L02-PD-1N cell lines,and specifically recognized PD-1 extracellular domain protein.Using tissue immunofluorescence technology,it was found that the aptamer P13 can bind to the PD-1 molecule on the surface of T cells in the tissues of tuberculosis and tumor patients.These indicate that the aptamer has the potential for application in disease diagnosis and treatment,which may provide a new avenue for TB and tumor disease control.