Preparation Of Recombinant PD-1 And PD-L1 Extracellular Fragments And Their Aptamers

BackgroundProgrammed death factor receptor 1(PD-1)is an important immune checkpoint in the body.The PD-1/PD-L1 pathway composed of PD-1 and PD-L1 is an important mechanism of immune cell failure,immune function damage and chronic viral infection.In addition,it is also closely related to the regulation of autoimmune diseases,graft immune rejection,abnormal genes,and pregnancy-related immune diseases.This has also made targeted PD-1,PD-L1 immunotherapy,or concomitant detection for this therapy a recent years Hot research.The aptamer selected by SELEX technology is used to diagnose and evaluate the disease by detecting biomarkers or to interfere with and block the pathway as a PD-1/PD-L1 inhibitor to promote the development of PD-1/PD-L1 targets in disease prevention Greater application value.ObjectiveThe soluble recombinant proteins of PD-1 and PD-L1 extracellular fragments were prepared,and the two were used as targets to select their respective highly specific and high affinity aptamers,and the screened aptamers were identified and characterized.Method(1)Construction of PD-1 and PD-L1 prokaryotic engineering bacteria using genetic engineering technology.Purified recombinant protein was obtained through fermentation,lysis,inclusion body treatment,gel column desalting,and separation chromatography.Tricine-SDS-PAGE,Western blot,peptide mass fingerprinting,and BCA assay were used to detect and analyze the recombinant protein.(2)A random ssDNA aptamer library was synthesized and a SELEX selecting protocol was established.Denaturing PAGE,qPCR,nucleic acid quantification,QCM and other methods were used to monitor the quality of the selected and enriched secondary libraries.(3)The final enriched product was TA cloned,sequenced,analyzed by related software,sequence information was intercepted,aptamers were synthesized,and aptamers were characterized by Dot blot and EMSA.Result(1)Using prokaryotic expression system,high-purity recombinant proteins of PD-1 and PD-L1 extracellular fragments were successfully prepared.(2)After 13 rounds of selecting with Amino Magnetic Beads-SELEX,the enrichment state reached saturation.Finally,27 aptamers were synthesized by molecular cloning and sequencing analysis.(3)27 aptamers were initially characterized by Dot blot and EMSA to obtain A-1 and A-6 aptamers for the PD-1 target protein,and B-10 aptamers for the PD-L1 target protein and predict their stem Ring secondary structure.ConclusionIn this study,A-1 and A-6 aptamers of the PD-1 target protein were successfully selected,and B-10 aptamers of the PD-L1 target protein were successfully developed.For the subsequent development of detection kits and targets for the PD-1/PD-L1 target,The foundation for nucleic acid drugs.