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Pathogenic Effect of Avian Leukosis Viruses in Chicken
Průková, Dana ; Geryk, Josef (advisor) ; Němečková, Šárka (referee) ; Grubhoffer, Libor (referee)
comparativestudyof pďhomorphologicalchangesin lymphoidtissuesinducedby ALVs of different subgroupspecificity, hasnot beenperformed.Usingthe chicken model we aimed at clarification of additional features,especially pathomorphologicaland immunological chara- cteristics of the wasting diseaseinducedby different strainsof ALV{. In orderto achievea propercompďison,alsoadditionalALV subgroup(A'B'D) wereincluded. on the otlrerhand, several retroviruseshave been found to induce kidney fumoÍs. MAv2 (N) is an avian nonacuteoncogenicÍetrovirusof subgroupB ALv. In chickens infec- ted in ovo or early afrerhatchingit induces,with high efÍrciency'multip|eclonal embryonic- type tumorsof kidney - nephnoblastomas(Wattsand Smith, 1980).The model of MAV2 (N)- induced chicken nephroblastomais basedon the assumedabiliý of MAV2(N) retrovirusto transformcells by insertionalmutagenesis,thatis, by a deregulationof expressionof geneshit by the proviral integration.This avian model ofnephroblastomarepresentsa valuabletool for identi$ing genesinvolved in themaligrant transformationof cells. It is assumedthatmacro- scopic nepfuoblastomasarise by clonal expansion of blastema cells in which MAV2 (N) provirus has deregulatedspecific genescontroling differenciation and proliferation @ajer et ď.' 2003). Summary ALVs have a large potential to...
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Study of the effect of immunological sdjuvants on experimental treatment of HPV-induced tumors by recombinant VACV and DNA vaccines
Gabriel, Pavel ; Němečková, Šárka (advisor) ; Mělková, Zora (referee) ; Reiniš, Milan (referee)
1 ABSTRACT The success of cancer vaccines depends on factors associated with the vaccine, which define the main parameters of effective immune responses such as its size and quality, as well as on factors related with the host, represented by the immunosuppressive mechanisms that allow the tumor to escape recognition by the immune system or negatively influence the function of effector T-cells. Attenuated, non-replicating viruses are at present preferred as VACV for safety reasons. A problem may arise concerning their lack of immunogenicity. Through the deletions of non-essential genes, vaccination vectors are therefore developed based on attenuated rVACV capable of replication, which induce a strong immune response. Genes of various immunological adjuvants (e.g., genes for cytokines and costimulatory molecules) are inserted into the vectors for the purpose of eliminating the influence of the immunosuppressive mechanisms of tumors. The first part of the work describes our study of the influence of vCCI on biological properties of rVACV derived from the Prague strain. Testing of vCCI deletion and insertion mutants expressing tumor associated protein HPV16 E7 has shown that secreted vCCI attenuated the virus in vivo, which correlated with reduced levels of the corresponding CC chemokines in the blood compared...
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Expression of sTGFbeta RII-Fc-Jun from recombinant vaccinia virus
Samková, Zuzana ; Němečková, Šárka (advisor) ; Španielová, Hana (referee)
Expression of sTGFbetaRII-Fc-Jun from recombinant vaccinia virus TGFß has a biphasic role in tumorigenesis. In early phases it acts as tumor sup-pressor. However, in late phases when cells have escaped selectively from the antimito-genic response of TGFß, it may act as a promoter of tumor progression and invasion. One way of control tumor formation and progression is blocking of TGFß signalling pathways in late phases of tumorigenesis. We have constructed recombinant vaccinia virus P13 expressing soluble TGFbeta type II receptor fused with the Fc fragment of IgG1 and with Jun fragment (sTbetaRII-Fc-Jun). This sTbetaRII-Fc-Jun is supposed to increase the effect of antitumor vaccinia virus vaccine expressing SigE7LAMP, which is investigated for the treatment of the HPV-16 associated cervical cancer. Binding of sTbetaRII-Fc-Jun to protein G were tested by SDS-PAGE and by im-munoblotting. We found that Jun fragment and sTbetaRII fragment do not block Fc bind-ing site for protein G. sTbetaRII-Fc-Jun was characterised using SDS-PAGE and immunoblot analysis. We observed that the amount of sTbetaRII-Fc-Jun was higher in cell supernatans of in-fected cells in comparison to cell lysates. In cell lysates we observed higher amount of sTbetaRII than sTbetaRII-Fc-Jun. The expression of sTbetaRII-Fc-Jun was stronger under...
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Studies of minor capsid proteins of the mouse polyomavirus
Vít, Ondřej ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Mouse polyomavirus (MPyV) is a small non-enveloped virus. Its capsid consists of 72 pentamers of the major capsid protein VP1. The central cavity of each VP1 pentamer contains one minor capsid protein, either VP2, or VP3. The minor capsid proteins are dispensable for capsid formation, but their presence is required for infection of the host cell, presumably because of their anticipated functions during virus entry. After internalization, MPyV virions traffic to endoplasmic reticulum (ER). VP2 and VP3 have been proposed to function as factors responsible for penetration of ER membranes, which is required for subsequent delivery of the viral DNA into the nucleus, a key step of the early phase of MPyV infection. Three hydrophobic domains were predicted in the sequence of VP2 and VP3. First in the unique Nterminal part of VP2, second and third in the common part of VP2 and VP3. The third domain corresponds to C-terminal VP1binding alpha-helix. It has been previously found in our laboratory, that VP2 and VP3 fused to N-terminus of EGFP, when expressed in mammalian cells, display properties similiar to the wild-type VP2 and VP3, namely affinity to intracellular membranes and high cytotoxicity. Expression plasmids carrying mutated VP2 and VP3 fused to Nterminus of EGFP were prepared to determine the hydrophobic...
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Preparation of monoclonal antibody against the major structural protein, VP1, of BK virus and construction of plasmids for protein interaction analysis by BiFC method.
Kozmanová, Alexandra ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
The aim of this study was to create a monoclonal antibody against VP1 protein BK virus for study of individual steps of its replication cycle. BK virus is human polyomavirus which causes serious disease - polyomavirus associated nephropathy in immunocompromised patients after renal transplantation or hemorrhagic cystitis after bone-marrow transplantation. Virions of BK virus have capsids with icosahedral symmetry consisting of 360 molecules of the major capsid protein VP1 and two minor structure proteins VP2 and VP3, which are not exposed on the capsid surface. VP1 is highly immunogenic what gives us possibility to produce antibodies capable of BK virus monitoring by capsid protein detection. Empty BK virus particles produced in insect cells from a recombinant baculovirus were used as antigen to immunize mice for preparing hybridomas. Cloning of hybridomas created by fusion of myeloma cells with B-lymphocytes from immunized mouse gave us series of primary hybridoma clones. One of them, 5/G10/A5/B2/8, was cloned to homogenity and monoclonal antibody against VP1 BK virus was purified from hybridomas medium by affinity chromatography. The analysis of antibody properties revealed that it recognizes a conformation epitope and can be used for VP1 protein and virion detection in cells by indirect...
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