神经生长因子低亲和力受体(p75NTR)的模拟配基的筛选

Phage Display Peptide Library Selection on Whole Cells Yields Peptides Specific for NGF Receptor p75NTR

人神经生长因子低亲和力受体 (p75NTR)转染R2细胞而建立的R2L1细胞 ,在去血清培养时发生凋亡 ,该作用可被神经生长因子 (NGF)所抑制 .用R2L1和R2两种细胞差式筛选噬菌体随机 7肽库和 1 2肽库 ,获得和p75NTR特异结合的噬菌体 .测定DNA序列后得到有关多肽的氨基酸序列 .7肽库共有序列为C (H D)LP(K M)HPM C ;1 2肽库优势序列为TLPSPLALLTVH .化学合成相应的 2个短肽 .用细胞结合法和ELISA方法证实阳性噬菌体和合成短肽能与p75NTR结合 。

R2L1 cell line has been established by transfecting rat brain cell line (R2 cell line) with human low affinity neurotrophin receptor p75NTR. The apoptosis of R2L1 cells cultured with depriving serum can be inhibited by nerve growth factor (NGF). Peptide 12 mer phage display library and peptide 7 mer phage display library were used to panning against R2L1 cell line and R2 cell line. After three rounds panning, two dominant sequences were obtained: TLPSPLALLTVH(Ph.D 12), C (H/D)LP(K/M)HPM C (Ph.D 7). Two peptides were synthesized: C HLPKHPM C and RTLPSPLALLTVHD. Two amino acids were added at two sides of 12 mer peptide in order to enhance its solubility. Tests demonstrated that the positive phages and the chemically synthesized peptides could bind p75NTR by using ELISA method and cell binding method. The positive phages and the mimic short peptides action on R2L1 apoptosis was observed from the following two aspects: the morphological changes of cells by optical microscopy; the sub G1 peak in flow cytometric analysis. All of them can inhibit the apoptosis of R2L1 cells cultured with depriving of serum.