PURPOSE: To screen mimic peptides of lipopolysaccharide binding protein(LBP) binding to CD14 from phage display random peptide library.
METHODS: Using CD14 as a target molecule and LBP as eluent, 4 rounds of biopanning of a phage random 12-mer peptide library were carried out. Then, 20 clones were selected randomly and used in binding test. Positive clones with high affinity were sequenced . The peptide was compared with LBP sequence , was synthesized, and was used to competitive inhibition test with LBP. Finaly, U937 cells were stimulated by Phorbol yristate acetate ( PMA ) to investigate the effects of the peptide on TNF-αexpression on U937 cells induced by LPS. The study was divided into five groups : control group , LPS group (100 ng/ mL LPS plus 100 ng/ mL rhLBP) , high dose peptide group ,middle dose peptide group , and low dose peptide group (1 000 ng/ mL ,100 ng/ mL , and 10 ng/ mL , respectively) . The concentration of TNF-αreleased from U937 cells was detected by ELISA. The mRNA of TNF-αwas determined by RT-PCR.
RESULTS: Out of the 20 clones, 16 were identified as positive. 8 of the 16 clones had more high affinity, and encoded a 12-mer peptide such as FHRWPTWPLPSP which was not homologous with the sequence of LBP. The peptide was a mimic peptide. It had high competitive inhibition with LBP in binding to CD14. TNF-αmRNA expressions in high and middle dose peptide group were lower than those in LPS group and higher than those in control group. The concentration of TNF-αin high and middle dose peptide group was lower than that in LPS group and higher than that in control group.
CONCLUSION: A mimic peptide of lipopolysaccharide binding protein binding (LBP) to CD14 is obtained by phage peptide library screening, and can reduce the release of TNF-αand the mRNA of TNF-α.
CLINICAL IMPLICATIONS: It has a potential protective effect on LPS induced inflammatory disorders such as acute lung injury.
DISCLOSURE: Guoming Wu, No Financial Disclosure Information; No Product/Research Disclosure Information