Screening Of Mimotpes Of The Humans Influenza A From Random Peptide Library Displayed On Phage

The highly contagious, acute respiratory illness known as influenza as appears to have afflicted humans. Humans have not found good therapy to influenza and still depend on vaccines. But the variation of influenza virus reduces the efficacy of the inactivated vaccines that is licensed for parenteral administration in humans. So it is urgent to find a novel influenza vaccine that is cross-protective to different subtypes. In this study, we isolated the mimotopes having cross-protection between H3N2 and HINI. 1. The affinity selection and verification of influenza virus mimotopesThe 12-mer phage library was amplified in E.coli XI-Blue, purified by PEG/NaCl solution and resuspended in TBS. The wells of ELISA plate were coated with pAb (100ng/μl) against H3N2, then phage was added to the wells. After incubation, the wells were washed vigorously with TBST to remove nonbinding phage. Phage bound to the antibody were eluted with 0.2mol/L Glycine-HCl(pH2.2) for 10 min at room temperature andneutrialized with 2mol/L tris-HCl(pH9.1). Titered and amplified the eluted phages. The amplified phage be used repeat the selection. The selection was repeat three times. The titter of phage indicated that the eluted phage enriched with the rounds increasing.Amplified in E.coli XI-Blue, the eluted phage in the third rounds was poured onto LB/IPTG/Xgal plate. We selected randomly 18 clones and amplified them, then confirmed positive clones by ELISA.We sequenced the positive clones. The result was that the amino acid sequences of 5 clones were same and were homologous with influenza virus protein FBI, PA, NS1, the amino acid sequences of two others were same and the amino acid sequences of the rest shared identity. The result indicated that we had selected the mimotopes of influenza virus A. 2. Assaying of the protective effect of mimotopesWe divided the mimotopes into three groups based on their amino acid sequences' homology, with phage Ml3 as the control group, then immunized 6-week BALB/c mice, 10 mice per group. After amplifing positive phages and quantification, we used them to immunizing mice subcutaneously at 2-week intervals. The dose given is 100ug phage per mouse at first immunization and 30ug phage per mouse at the latter two immunizations. Bleeds were taken from all mice 2 weeks after each immunization. Then we assayed the influenza virus specific antibody and the ratio of IgG1 to IgG2a in mice serum. The rates of inducing specific antibody of four groups mice were 72.7%, 25.0%, 33.3%, 0% (control group) after second immunization and 90.9%, 88.9%, 85.7%, 0% (controlgroup) after third immunization. The result indicated that the mimotopes could induce the specific antibody against influenza virus. And the quantity of IgGl was much more than the one of IgG2a in mice serum, so we deduced initially that the main type of immune reaction that the mimotopes induced was cell-mediated immune response.The A/PR/8/34(HlN1) adapted to mouse lung was propagated in allantoic cavity of 9-day-old embryonated chicken eggs and titered by hemagglutinin test, and the tilers was 1:320. Diluting virus with 0.85% NaCl into concentration of 10-1-108, the normal mice were challenged by intranasal inoculation of a range of 200ul virus dilution to assay its LD50. At the result, the LD50 was 10-3.625. Two weeks after the third immunization, the mice were challenged with the 5xLD50 A/PR/8/34 and monitored over the following 14 days. The survival rates of four groups were 40%, 20%, 40% and 0%(control group). And the survival rate of the control group is smaller than the ones of immunizing groups. The result indicated that the mimotopes could induce part crossed protection against H1N1.In conclusion, we acquired the cross-protective mimotopes of influenza virus A and explored a new way to reseach for the cross-protective epiptops.