| Hantaviruses cause two kinds of disease of natural focus, hemorrhagic feverwith renal syndrome (HFRS) and Hantavirus pulmonary syndrome (HPS). Thereare about50,000to100,000cases of HFRS/HPS occurred every year throughoutthe world. The mortality rate varies from0.1%to40%according to thedifference in virus type. During the period of1950to2010there were a total of1,600,000cases of HFRS reported in China, which makes China as the mostendemic country worldwide. The HFRS is prevalent usually in rural areas wherethe patients are poorly diagnosed and treated in the local hospital, which makesit hard to collect enough and complete samples to perform clinical scientificresearch. Currently, there are no specific therapeutic medicine and regimenavailable for HFRS, although ribavirin and interferon-α are often used asantiviral drugs in treatment of HFRS. However, ribavirin has been not approvedby the Food and Drug Administration (FDA) to treat HFRS, and the study of the effect of ribavirin for HFRS is still conducted in Seoul, Korea, which now goesto phase II and will be completed in February,2013. Thus, it is an urgent task toelucidate the pathogenesis of the HFRS and to conduct research for the potentialantiviral drugs for HFRS.Antiviral peptides are new bioactive molecules which have multi-functionalroles in the experiments for treatment of cancers or infectious diseases. However,the work of screening the functional peptide is usually costly and time-consuming. Phage-display technique is a suitable and powerful method forselecting peptides, proteins or antibodies with affinity and specificity to amolecule or protein of interest, and it is also an economic way to screen antiviralpeptides.The process of viral infection contains two stages: attachment and penetration.Viruses attach to the target cells by the way of binding ligands to the receptorson cellular surface. Recent studies have been proved that the β3integrin is oneof the key receptors of hantavirus infection and blockade of β3integrin couldinhibit hantavirus infection by affecting virus entry into the target cells. In thepresent study, phage-display technology was used to screen peptides which canspecifically bind to β3integrin for eight rounds. After selection andamplification processes of the phage library on β3integrin, the screenedpeptides on the surface of the phage were sequenced and alignments of eachpeptide were conducted to get the biological information. Three of the screenedpeptides were synthesized and tested for their ability to inhibit hantavirusinfection Further studies are needed to optimize and examine the inhibitoryability of these peptides.Results:1. The optimism of the bacteria propagation The E.coli (strain ER2738) was used to propagate phages and titrate thephage clones. To propagate phages, the optimal time of phage infection is2.5h,while the optimal time for titering phage stock solution with bacteria is5.5h.2. The panning of the phage libraryWe conducted eight rounds of panning of the phage display libraryexpressing cysteine-constrained heptapeptides. The amount of the β3integrinwas gradually decreased, while the washing time was increased in order to getthe phage clones with high avidity and specificity with β3integrin. The titers ofevery round of phage clones were measured by the method of plaque titration.The results showed that the titer of phage clones was increased with the roundsof panning process, indicating that the highly bioactive phages were gathered.3. Sequences of phage DNAEighty clones of phage plaques were collected and amplified. The peptidesequences of individual plaques were deduced by sequencing the phage genomethat encoded them. Five peptides which had the highest frequencies ofappearance were finally identified (TGVKGPG, LPLTPLP, KLTSSPT,SPVGPLP and DHRNHLV). Sequence alignments of these five peptides toHantaan glycoproteins were done in a pairwise fashion. The results showed thatthere were significant similarities among these peptides and Hantaan virusglycoprotein.Conclusion: We screened and identified several novel peptides which maypotentially inhibit the entry by hantaan virus into target cells via β3interginthrough the method of phage display technology. |
