The Screening Design And Primary Functional Mechanism Research On Anatgonist Peptide Of Broad-spectrum Chemokine Receptor Encoded By Virus

Objective In this study, based on the US28 unique combination of broad-spectrum chemokine receptor activity and herpes-like virus common recognition features, using affinity selection, biological activity and pharmacodynamics of anti-virus tools, get positive clones and to clarify the mechanism and effect, to study from US28 with chemokines block the binding of antagonists to the treatment of human cytomegalovirus to provide new drugs, while broad-spectrum anti-viral drugs lay the technical foundation design.Method Using TRANSWELL chemotaxis and chemotactic suppression experiment, test screening lead compounds that target molecules H22 activity; selected 25 kinds of 4 full sequence of Construction of chemokine expression of peptide libraries, as a screening platform, the use of phage display panning, and H22 binding affinity and sequence; Fmoc-solid phase synthesis of cloned sequence; by calcium phosphate transfection techniques to the virus supernatant HCMV US28 gene was amplified as a template to construct stable expression of HCMV US28 gene in NIH/3T3 cell lines, as antagonist pharmacodynamics research platform; and then cross-linking experiments to verify the inhibitor peptide and receptor binding, measured by flow cytometry to determine the calcium ion concentration on the receptor peptide induced intracellular signaling to the block Finally, experiments using anti-viral peptide confirmed the antagonistic effect of the virus.Results Chemotaxis experiments show that peptides do not stimulate but inhibit cell directed chemotactic chemokines by a variety of human induced directional migration, build successful expression of peptide libraries can be screened and 24 positive clones were selected of which 12 were sequenced by 8 sequence, of which eight clones expressed LNAHCAL conservative sequence; through the pcDNA3.1 eukaryotic expression vector was successfully constructed and stable expression of US28 in the NIH/3T3 cell line, ligand receptor cross-linking experiments showed that, H9 may be a good membrane receptor US28 combination of functional cell experiments showed that H9 can be reduced by a physiological chemotactic factor receptor-induced calcium ion concentration in itself did not cause changes in calcium ion concentration, anti-virus function experiments showed that H9 can inhibit the induction of HCMV AD 169 of plaque formation, reduce disease virus infected cells and inhibition of virus replication in cells of infections caused by inhibition of HCMV AD 169 CEP's EC50=0.46ng/ml, the role of inhibition of pp65 antigens EC50=0.34ng/ml.Conclusion H22 filter material target molecules can significantly block the chemotactic measurement-based activities through the construction of peptide libraries can be screened to clone sequences LNAHCAL, screening methods feasible; positive clones can mimic inhibitory peptide H9 cells exogenous rational combination of chemokine and US28, has blocked the effect of receptor signal transduction in vitro with anti-HCMV AD 169 activity, can inhibit the virus from entering cells and intracellular replication, inflammation and virus can be as broad-spectrum chemokine antagonist, for research related to virus prevention work them.