论文部分内容阅读
Objective: One hallmark of cells entering the apoptosis was the phosphatidylserine (PS) externalization from the cytoplasmic to the extracellular side of the membrane.Annexin V bound selectively to membrane PS residues with nanomolar affinity and was utilized to image PS exposure with radioactive isotopes.The drawback of the annexin V is the significant uptake in nontarget tissues.The solution to this problem is developing small molecule such as peptides.The aim of this study was to screen peptides binding to phosphatidylserine for apoptotic/dead cell imaging.Methods: The phosphatidylserine was coated on immuno-absorbant plates.The linear 7-mer,12-mer, cyclic 7-mer peptide phage display libraries were added to plates to bind phosphatidylserine.After three rounds of absorption-elution-amplification for biopanning and enrichment, the stronger binders to phosphatidylserine from peptide phage display libraries were obtained in one pot.Thirty-two phage clones were isolated for proliferation and their individual specificities against phosphatidylserine were tested by enzyme linked immuno-sorbant assay (ELISA).The peptide-displayed phages with better specificities were chosen for single-strand DNA extraction and sequencing.The amino acids sequences of phosphatidylsefine-avid peptide were deduced from DNA sequence by triple codon degeneration.Competitive enzyme-linked immunosorbent assay (ELISA) with annexin V and synthetic peptide was performed to confirm the specificity of the selected peptide-carried phages.The phages were conjugated with hydrazinonicotinyl (Hynic) and labeled with radioactive isotope 99mTc to test their binding specificity to PS-exposed erythrocytes.Results: Forteen peptides specifically against phosphatidylserine were screened out from the linear 7-mer, 12-mer, cyclic 7-mer peptide phage display libraries separately.Competitive ELISA with annexin V and synthetic peptide confirmed the specificity of the selected 12-mer peptide-carried phages.The 99mTc-labeledphages bound PS-exposed erythrocytes specifically.Conclusions: The phosphatidylserine-avid peptides were screened out from peptide phage libraries by in vitro biopanning, which have paved a way for apoptotic/dead cell imaging.