Screening And Identification Of H5N1 Virus Hemagglutinin Neutralizing Antibody And Epitope Identification And Sorrel Interaction Protein

Partâ… Identification of Epitopes for Neutralizing Monoclonal Antibodies against Highly Pathogenic Avian Influenza H5N1Virus HemagglutininSustained and widespread highly pathogenic avian influenza (HPAI) H5N1epidemics represent a significant public health hazard in China, not only because they cause mortality in poultry, but also because they increase the risk of a human influenza epidemic. Numerous attentions have been paid to the first case of human infection avian influenza of Hongkong in1997, suggesting that the species barrier had been broken, in another world, human could be infected directly. At present, there is no definite evidence that avian influenza can transmit from person to person, but there is a report that H5N1could be transmitted between ferrets by natural evolution or individual amino acid mutation.H5N1viruses infect host cells through the binding of surface hemagglutinins and the receptors on target cells and hence the hemagglutinins have become the major antigens for the generation of antibodies against H5N1viruses. As a significant role involved in the fight of immune system against H5N1viruses, neutralizing antibodies are able to block virus transmission and eliminate infected cells. However, the generation of neutralizing antibodies and application of clinical treatment largely depend on the identification of neutralizing antigenic epitopes of HA.In our previous studies, four HA genes, namely A/Hongkong/213/03H5N1HA(HK-HA), A/Anhui/01/05H5N1HA(AH-HA), A/Xinjiang/01/06H5N1HA(XJ-HA) and A/Bar-headed Goose/Qinghai/1/05H5N1HA(QH-HA), isolated from avian and human cases, were systemically analyzed with epidemiological and molecular biological methods and confirm that these four HA genes were the dominating epidemic subsets of H5N1viruses in China. We also identified six neutralizing epitope candidates of HA proteins.To further validate these neutralizing antigenic epitopes of HA, six candidate peptides were fused to tandem epitopes for the generation of monoclonal antibodies using a patent technology of SEAL (Surface Epitope Antibody Library) to screen neutralizing monoclonal antibodies against individual epitope on the level of H5N1 pseudotyped virus and recombinant HA proteins.In the present study, six linear epitopes were assembled randomly into four poly-epitope peptides joined by linkers. DNA fragments encoding the designed poly-epitope peptides were obtained by overlapping extension polymerase chain reaction or de novo DNA synthesis, and cloned into a prokaryotic expression vector with an immunogenicity enhancer. The poly-epitope peptides were over-expressed in E.coli, and purified by Ni-affinity chromatography. At the same time, we generated five pseudoviruses. One advantage of pseudoviruses is its safety because it can only undergo a single-round of infection and its backbone is derived from the standard HIV-1in which the envelope gene is defective and owns an insertion of luciferase gene for monitoring viral infection. Neutralizing monoclonal antibodies were characterized by ELISA, Western blot and microneutralization assays.23hybridoma clones secreting anti-HA epitope monoclonal antibodies were obtained. Three IgG3subtype clones of them showed efficient neutralizing activities to H5N1pseudoviruses and could recognize rHAs from four different H5N1viruses. Furthermore, these three neutralizing monoclonal antibodies recognized respective epitopes in a dose dependent manner. Two important neutralizing epitopes positions of144-153and154-163were identified, which might serve as strong potential candidates for the development of an effective vaccine to protect human from highly pathogenic avian influenza H5N1virus in China. Part IIScreening and Identification of Thorase Interacting ProteinsIn this part, our research focused on the interaction proteins of a novel neuroprotective protein which we named Thorase, the Norse God of Thunder and Lightening. Thorase belongs to an AAA+ATPase family that was discovered by a strategy of functional screen.In our previous study, protein CHIP (17K) assay was performed to identify Thorase-interacting network.375proteins were identified as candidates of Thorase-interaction proteins. Then we focused on37interesting proteins and constructed a panel of pEGFP-tagged gene expression vectors by GatewayTM conversion system. GFP-tagged expression vectors were co-transfected with Thorase expression vectors into HEK293Tcells. Co-immunoprecipitation assay was performed and confirmed11Thorase-interacting proteins. The findings may lay the foundation for further investigating the biological functions of Thorase.