Rozprawy doktorskie na temat „EPITOPING”

Background: Novel peptides with epitopic activity can be discovered by a combination of peptide arrays and computational interpolation. These peptides can mimic the activity of cellular receptors or their ligands, and are thus called peptidomimetics. The activity of peptidomimetics evidently varies in accordance with the attributes of the peptide. The fibrin-mimetic, Arginine-Glycine-Aspartate (RGD), is a well known anti-thrombotic peptide which inhibits the interaction between the platelet integrin GPITbIIIa and fibrinogenlfibrin. Short peptides often suffer from high inhibitory concentrations in vitro and short clearance times in vivo. To increase vascular residence times of such antithrombotic peptides, they were coupled to macromolecular carriers. Hyperbranched polyglycerols (I{PG), macromolecules designed as a dendritic carrier species, at a range of HPG molecular weights (MW) were tested as the carriers for antithrombotic peptidomimetics. Methods: HPG of MW 3 to 500 kDa were conjugated with RGD at a range of substitution ratios. The optimum molecular weight and substitution ratio were then applied to the peptide SHAYIGLKDR, a vWf mimic peptide discovered through the use of bioinformatics. For peptidomimetics (RGD and SHAYIGLKDR), enzymatic proteolysis was employed to distinguish the specificity of the inhibitory activity among HPG, peptide, and the HPG-peptide conjugate. Flow cytometry, UV spectroscopy, compound light microscopy, and lummiaggregometry were used to characterize the function of the conjugates in vitro. Results: Conjugation of RGD to HPG resulted in a decrease of the inhibitory concentration required to interrupt platelet-fibrinogen interactions by up to three orders of magnitude. Inhibitory activity was directly related to the number of peptides attached per HPG. Similar results were found when high molecular weight HPG, selected from the RGD-related experiments, was used to carry the peptide SHAYIGLKDR. None of HPG, RGD, SHAYIGLKDR, or their conjugates caused spontaneous platelet activation, or inhibited thrombin-mediated platelet activation, showing that the peptides’ activity is directed specifically toward their targets: GPllblllalfibrinogen (RGD) and GPIb/vWf (SHAYIGLKDR) interactions. Tryptic digestion of conjugates confirmed that the inhibitory activity of HPG conjugates was dependent on the presence of the intact peptides. Conclusions: Conjugation of peptidomimetics or other molecules to macromolecular platforms such as HPG is a viable method to enhance the peptidomimetics’ activity. The degree of enhancement is dependent upon the level of peptide substitution as well as the size of the carrier.

La costruzione di un vaccino protettivo basato su peptidi sintetici di HIV è limitata dalla loro scarsa immunogenicità. Sfruttando le note proprietà immunomodulatorie del BCG e a seguito dell’identificazione in una sub-popolazione africana (Burkina Faso, Costa d’Avorio, West-Camerun) di epitopi virali in grado di suscitare una risposta immunitaria, abbiamo effettuato uno studio per verificare l’induzione di una risposta anticorpale e cellulare verso tali epitopi. Per prima cosa abbiamo valutato l’eventuale tossicità dei prodotti utilizzati e ci siamo chiesti se la presenza di peptidi di HIV nella cosomministrazione o nel costrutto ricombinante potessero influenzare la risposta verso lo stesso BCG. Le capacità adiuvanti del BCG wild-type e ricombinante sono state valutate sia nel modello murino che in primati non umani con il fine ultimo di formulare un vaccino pediatrico contro la trasmissione verticale dalla madre al figlio del virus di HIV attraverso il latte materno.
The formulation of a protective anti-HIV vaccine based on the used of sintetic HIV peptides is limited by their poor immunogenicity. Taking advantage of known immunomodulatory properties of BCG and identified immunogenic viral epitopes in an African sub-population (Burkina Faso, Ivory Coast and West-Camerun), we verified the induction of humoral and cellular response towards these epitopes. First of all we evaluated the potential toxicity of the product used by checking if the contemporary administration of these peptides with BCG or the administration of an HIV-peptide recombinant BCG could interfere with immunological response to BCG. We evaluated the adjuvant capabilities of wild-type BCG and of recombinant BCG both in a mouse and in non-human primate models to generate a pediatric vaccine against mother to child transmission (MTCT) of HIV virus through breast feeding.

L’attivazione immunitaria cronica, che caratterizza le malattie autoimmuni e le patologie infettive, è sostenuta da una persistente iperattivazione del sistema immunitario, da forte infiammazione dei tessuti colpiti dalla malattia e da apoptosi massiva delle cellule degli stessi tessuti. Nel corso di infezione da HIV (o da SIV) questi tre aspetti coesistono.Nell’infezione da HIV è dimostrata, in particolare, l’aumentata apoptosi, spontanea o indotta dall’attivazione, delle cellule T CD4+ e CD8+ ,ma in particolare delle cellule T CD4+ HIV-1 specifiche. I fenomeni alla base dell’attivazione immunitaria cronica nell’infezione da HIV potrebbero contribuire al mantenimento, quindi alla cronicità, dell’infezione stessa. Nel nostro laboratorio erano stati analizzati i profili proteici di cloni di linfociti T vivi o apoptotici, osservando che alcune proteine (tra cui proteine del citosheletro) vengono frammentate durante l’apoptosi. Era stato anche osservato che le caspasi, oltre ad essere necessarie, come era già noto, per la frammentazione delle proteine durante l’apoptosi, lo erano anche nel fenomeno della cross-presentazione delle cellule apoptotiche da parte delle DCs ai linfociti T CD8+ specifici. Ipotizzando che le proteine identificate possano essere immunogeniche, abbiamo sintetizzato un pannello di peptidi, ristretti per HLA-A2, derivati dalle proteine frammentate durante l’apoptosi e li abbiamo utilizzati in test Elispot. Nei pazienti testati esiste un ampio repertorio di linfociti T efCD8+ IFN-gamma+ specifici per diversi peptidi self. Le frequenze delle cellule T CD8+ effettrici specifiche per epitopi apoptotici correlano con la frequenza delle cellule T CD4+ apoptotiche. La citofluorimetria ha confermato l’esistenza, in vivo, di cellule T CD8+ specifiche per epitopi self apoptotici. Il fenotipo effettore di queste cellule è stato confermato dall’espressione di perforina e granzimi in una notevole porzione di cellule fresche CD8+ pentamero+ in tutti i soggetti infettati studiati. Le cellule T CD8+ pentamero+ da soggetti infettati da HIV producevano IFN-gamma , ex vivo, se stimolate con DCs che erano state pulsate con cellule apoptotiche derivate da cloni di cellule T CD95+. Questi dati indicano che, nel repertorio periferico degli individui infettati da HIV, in vivo, sono presenti cellule T efCD8+ IFN-gamma+ specifiche per gli epitopi derivati dal processamento delle proteine apoptotiche.
Chronic immune activation, that characterizes chronic viral or autoimmune diseases, is supported by a persistent hyperactivation of the immune system, by a strong inflammation of the tissues damaged and by a massive apoptosis of the cells in these tissues. During HIV (or SIV) infection these elements co-exist. In particular, it was demonstrated that the spontaneous or activation-induced apoptosis of CD4+ and CD8+ T cells (in particular of CD4+ HIV-1 specific T cells) increases in HIV infection. The phenomena that characterize the chronic immune activation in HIV infection may contribute to the maintenance of the same infection. In our laboratory, we analyzed the protein patterns of live or apoptotic T cell clones, observing that some proteins (for example cytoskeletal proteins) are fragmented during apoptosis. These protein fragments are generated by caspases. We observed that caspases are also important in the cross-presentation of apoptotic cells by DCs to specific CD8+ T cells. Assuming that the identified proteins can be immunogenic, we synthetized a panel of peptides, HLA-A2-restricted, derived from protein fragmentation during apoptosis and we used them in the Elispot test. In the tested patients there is a wide repertoire of efCD8+ IFN-gamma+ T lymphocytes specific to different self peptides. The frequencies of effector CD8+ T cells specific for apoptotic self epitopes are directly correlated with the percentage of apoptotic annexin V+ CD4+ T cells. FACS analysis confirmed, in vivo, the existence of CD8+T cells specific for apoptotic self epitopes. The effector phenotype of these cells was confirmed by perforin and granzyme expression in a considerable proportion of fresh pentamer+CD8+ T cells from all infected subjects studied. Pentamer+ CD8+ T cells from HIV-infected subjects produced IFN-gamma, ex vivo, after stimulation with DCs that had been pulsed with apoptotic cells derived from CD95+ T cell clones. These data indicate that efCD8+ IFN-gamma+ T cells, specific for naturally processed epitopes derived from apoptotic proteins, are present in the peripheral repertoire of HIV-infected individuals in vivo.

Certains adénovirus humains (HAdV) comme le sérotype 3 (appartenant au sous-groupe B) sont capables de former des particules pseudo-virales composées des deux protéines impliquées dans l’entrée virale : la base du penton et la fibre (= penton). En effet, 12 pentons sont capables de s’auto-assembler de manière symétrique pour former des particules appelées dodécaèdres (Dd). Dans le présent travail, nous avons modifié et caractérisé les dodécaèdres bases (c’est à dire des Dds sans fibres) de l’HAdV3 afin d’en faire une plateforme vectorielle multi-épitopique versatile appelée ADDomer (ADenovirus Dodecamer). Pour cela, nous avons identifié des régions de la base du penton permettant l’insertion de peptides d’intérêt et créé une plateforme génétique générique permettant l’insertion facile de ceux-ci par biologie synthétique. L’insertion de séquences codant un peptide d’intérêt directement dans le gène de l’ADDomer, résulte dans son exposition de manière multivalente à la surface de la VLP du fait de la pentamérisation puis de la dodécamérisation de la base. L’ADDomer a été produit et caractérisé afin d’évaluer sa capacité à vectoriser des épitopes linéaires ou structuralement complexes. Nous avons ensuite conçu une deuxième stratégie de vectorisation, toujours basée sur l’ADDomer mais cette fois-ci en utilisant l’interaction base/fibre. Un peptide mimant la partie de la fibre de l’HAdV3 (les 20 résidus N-terminaux) interagissant avec la base du penton a été élaboré pour servir d’adaptateur formant des liaisons covalentes avec l’ADDomer.Le comportement de l’ADDomer in vivo a été étudié dans un contexte vaccinal. Pour cela, nous avons injecté l’ADDomer chez la souris afin de valider son transport vers le système lymphatique. Nous avons également démontré que l’ADDomer était capable de s’internaliser dans les monocytes et dans des cellules dendritiques dérivées de monocytes et d’induire les caractères spécifiques de maturation de ces dernières. Fort de ces résultats, nous avons généré un ADDomer vectorisant un épitope du virus Chikungunya décrit pour être la cible d’anticorps neutralisants de patients infectés par ce virus. Pour finir cette étude in vivo, nous avons évalué la capacité de l’ADDomer-TevChik à induire la réponse anti-épitopique et nous avons ainsi démontré que la façon dont l’épitope est présenté à la surface de l’ADDomer était importante pour obtenir une réponse significative
Some human adenoviruses (HAdV) such as adenovirus derived from serotype 3 (belonging to subgroup B) are able to form virus-like particles composed of the two proteins involved in viral entry: the penton base and the fiber (= penton). Indeed, 12 pentons are able to self-assemble in a symmetrical manner to form penton dodecahedron (PtDd). In the present work, we modified and characterized the base dodecahedron (BsDd = PtDd without fiber) of HAdV3 in order to create a versatile multi-epitopic platform named ADDomer (ADenovirus Dodecamer). We have created a genetic platform allowing easy insertion of epitope(s) of interest (s) thanks to synthetic biology. The insertion of sequences encoding a peptide of interest in the ADDomer gene enable a multivalent exposure at the surface of the VLP due to the pentamerization then to the dodecamerization of the penton base. ADDomer has been produced and characterized to assess its ability to vectorize linear or structurally complex epitopes. We then designed a second vectorization strategy, still based on the ADDomer, but using the interaction penton base / fibre. A peptide mimicking the part of the Ad3 fiber interacting with the penton base (the 20 N-terminal residues) has been designed to serve as an adaptor forming covalent bonds with the ADDomer.The behavior of the ADDomer in vivo has been studied in a vaccine context. For this, we injected the ADDomer in mice to validate its transport to the lymphatic system. We have also demonstrated that ADDomer is able to internalize monocytes and dendritic cells derived from monocytes (MoDC) and induces the specific characters of MoDC maturation. Based on these results, we generated an ADDomer vectorizing an epitope of the Chikungunya virus (ADDomer TevChik) described to be the target of neutralizing antibodies of patients who have been infected by this virus. To conclude this in vivo study, we assessed the ability of ADDomer TevChik to induce the anti-epitopic response and thus demonstrated that the way the epitope is displayed on the surface of the ADDomer was important to obtain a meaningful response

Ng Wai-yan.
Thesis submitted in: September 2003.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 200-225).
Abstracts in English and Chinese.
Statement --- p.iii
Acknowledgments --- p.iv
Abbreviations --- p.v
Abstract --- p.vii
Abstract (Chinese version) --- p.ix
Table of contents --- p.xi
List of Figure --- p.xx
List of Table --- p.xxiii
Chapter Chapter 1 --- : General Introduction --- p.1
Chapter 1.1 --- Toxoplasma gondii and Toxoplasmosis --- p.1
Chapter 1.1.1 --- Biology and life cycle of Toxoplasma gondii --- p.2
Chapter 1.2 --- Treatment of Toxoplasmosis --- p.4
Chapter 1.2.1 --- Chemotherapy --- p.4
Chapter (a) --- Pyrimethamine and sulfadiazine --- p.4
Chapter (b) --- Clindamycin and Spiramycin --- p.4
Chapter (c) --- Hydroxynaphthoquinones and new Macrolides --- p.5
Chapter 1.2.2 --- Toxoplamsa Vaccine --- p.5
Chapter (a) --- Vaccine using mutant strain of T. gondii --- p.6
Chapter (b) --- Subunit vaccine --- p.6
Chapter 1.3 --- The Immune Responses --- p.8
Chapter 1.3.1 --- Protective immune responses --- p.8
Chapter (a) --- Cytokines involved in the immunity against T. gondii --- p.10
IFN-γ: Role in resistance to infection --- p.10
TNF-α: Synergetic Role with IFN-γ --- p.10
IL-12: Role in T cell differentiation --- p.11
IL-10: Role in immuno-regulation --- p.12
Other cytokines --- p.12
Chapter (b) --- Immunomodulatory role of Nitric oxide (NO) --- p.13
Chapter (c) --- Humoral immunity against T. gondii --- p.13
Chapter 1.3.2 --- Toxoplasma proteins known to elicit T cell-dependent immunity --- p.14
Chapter (a) --- Surface antigen 1 (SAG1/ P30) --- p.16
Chapter (b) --- Dense granules protein 2 (GRA2/ P28) --- p.17
Chapter (c) --- Rhoptry protein 2 (ROP2/ P54) --- p.18
Chapter 1.3.3 --- Appropriate vaccine design --- p.18
Chapter 1.4 --- Aim of the study --- p.23
Chapter Chapter 2 ： --- Expression of the Synthetic Gene Encoding the Multi-epitopic Antigen (MEA) for Toxoplasma gondii in Escherichia coli --- p.25
Chapter 2.1 --- Introduction --- p.25
Chapter 2.1.1 --- Multi-epitopic antigen for T. gondii as vaccine --- p.26
Chapter a) --- "Epitopes from Toxoplasma antigens, P30, GRA2 and ROP2" --- p.26
"T-cell epitopes from P30 (aa 138-154, aa 191-209)" --- p.26
T-cell epitope from ROP2 (aa 197-216) --- p.27
T-and B-cell epitope from GRA2 (aa 171-185) --- p.27
Chapter b) --- Helper T-cell epitope from tetanus toxin --- p.27
Chapter c) --- Linker --- p.28
Chapter 2.1.2 --- Escherichia coli Expression System --- p.31
Chapter (a) --- The T7 RNA polymerase and T7 lac Promoter --- p.31
Chapter (b) --- Other translational elements --- p.32
Chapter (c) --- Fusion partner --- p.33
Chapter (d) --- Choice of expression host strain --- p.33
Chapter 2.2 --- Materials --- p.35
Chapter 2.2.1 --- Bacterial strains --- p.35
Chapter 2.2.2 --- Mouse strains --- p.35
Chapter 2.2.3 --- Chemicals --- p.35
Chapter 2.2.4 --- Nucleic acids --- p.36
Chapter 2.2.5 --- Kits and reagents --- p.37
Chapter 2.2.6 --- Antibodies --- p.37
Chapter 2.2.7 --- Solutions --- p.38
Chapter 2.2.8 --- Enzymes --- p.40
Chapter 2.2.9 --- Primers --- p.40
Chapter 2.3 --- Methods --- p.41
Chapter 2.3.1 --- Design and synthesis of the synthetic gene encoding the multi-epitopic antigen for Toxoplasma gondii --- p.41
Chapter 2.3.2 --- Cloning of the MEA into the E. coli expression vector --- p.41
Chapter (a) --- Preparation of plasmid : pCRII-TOPO-MEA and pET30a+ --- p.43
Chapter (b) --- PCR amplification of MEA from pCRII-TOPO-MEA --- p.44
Chapter (c) --- Digestion and purification of the E. coli expression vector pET30a+ with Ncol --- p.44
Chapter (d) --- Fill-in Ncol cut pET30a+ --- p.44
Chapter (e) --- Purification of DNA fragment from agarose gel --- p.45
Chapter (f) --- "Ligation of MEA fragment and Ncol cut, fill-in pET30a+" --- p.45
Chapter (g) --- Preparation of DH5a competent cells --- p.46
Chapter (h) --- Transformation of recombinant pET30a+-MEA --- p.46
Chapter (i) --- PCR screening and plasmid preparation for the putative pET30a+- HisMEA --- p.46
Chapter (j) --- Cycle sequencing reaction on putative plasmid pET30a+-MEA --- p.47
Chapter 2.3.3 --- Expression and purification of his-tag MEA --- p.48
Chapter (a) --- Expression profile of His-tag MEA production by IPTG induction --- p.48
Chapter (b) --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.49
Chapter (c) --- Estimation of His-tag MEA production in induced bacterial lysate --- p.50
Chapter (d) --- Purification of His-tag MEA --- p.50
Chapter (e) --- Braford Protein Microassay (Bio-rad) --- p.50
Chapter 2.3.4 --- Characterization of his-tag MEA --- p.51
Chapter (a) --- Western blot of induced bacteriallysate by monoclonal anti-his-tag antibody --- p.52
Chapter (b) --- N'-terminal amino acid sequencing of His-tag MEA --- p.52
Chapter (c) --- Western blot of His-tag MEA with seropositive serum of mice and human --- p.53
Chapter (d) --- Preparation of Toxoplasma gondii lysate --- p.54
Chapter (e) --- Western blot of purified his-tag MEA and T. gondii Lysate by anti- serum of recombinant his-tag MEA --- p.54
Chapter 2.4 --- Results --- p.56
Chapter 2.4.1 --- Design and synthesis of the synthetic gene encoding the multi- epitopic antigen for Toxoplasma gondii --- p.56
Chapter 2.4.2 --- Cloning of MEA into E. coli expression vector --- p.59
Chapter 2.4.3 --- Expression and purification of His-tag MEA --- p.61
Chapter 2.4.4 --- Charterization of His-tag MEA --- p.67
Chapter 2.5 --- Discussion --- p.73
Chapter 2.5.1 --- Design and synthesis of the synthetic gene encoding the multi-epitopic antigen for Toxoplasma gondii --- p.73
Chapter 2.5.2 --- Cloning of MEA into E. coli expression vector --- p.77
Chapter 2.5.3 --- Expression and purification of His-tag MEA --- p.77
Chapter 2.5.3 --- Characterization of His-tag MEA --- p.78
Chapter Chapter 3: --- Immunological Studies of the Multi-epitopic Antigen (MEA) for Toxoplasma gondii in Mouse Models --- p.82
Chapter 3.1 --- Introduction --- p.82
Chapter 3.1.1 --- "Expression of P30, GRA2 and ROP2 in E. coli expression system" --- p.82
Chapter (a) --- Expression of P30 --- p.82
Chapter (b) --- Expression of ROP2 --- p.83
Chapter (c) --- Expression of GRA2 --- p.84
Chapter 3.1.2 --- Immunizations --- p.84
Chapter (a) --- Choices of animals --- p.84
Chapter (b) --- Adjuvants --- p.85
Chapter 3.1.3 --- Measurements of cellular immune responses in vaccinated mice --- p.86
Chapter 3.2 --- Materials --- p.87
Chapter 3.2.1 --- Mouse strains --- p.87
Chapter 3.2.2 --- Chemicals --- p.87
Chapter 3.2.3 --- "Culture Medium, buffer and other solutions" --- p.87
Chapter 3.2.4 --- Nucleic acids --- p.87
Chapter 3.2.5 --- Kits and reagents --- p.88
Chapter 3.2.6 --- Solutions --- p.88
Chapter 3.2.7 --- Enzymes --- p.88
Chapter 3.2.8 --- Primers --- p.89
Chapter 3.3 --- Methods --- p.90
Chapter 3.3.1 --- "Construction of pET-P30, pET-GRA2 and pET-ROP2" --- p.90
Chapter (a) --- Construction of pET-GRA2 --- p.93
Chapter (i) --- Preparation of total RNA from tachyzoite of T. gondii (RH strain) --- p.93
Chapter (ii) --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.93
Chapter (iii) --- Cloning of pET-GRA2 --- p.94
Chapter (b) --- Construction of pET-ROP2 --- p.95
Chapter 3.3.2 --- "Expression and characterization of his-tag P30, his-tag GRA2 and his-tag ROP2" --- p.95
Chapter (a) --- "Expression profile of his-tag P30, his-tag ROP2 and his-tag GRA2" --- p.96
Chapter (b) --- Western blot of induced bacterial lysate with mono-clonal anti-his-tag antibody --- p.96
Chapter (c) --- Western blot of induced bacterial lysate with anti-serum against his-tag MEA --- p.97
Chapter (d) --- "Purification of his-tag P30, his-tag GRA2 and his-tag ROP2 from induced bacterial lysate" --- p.97
Chapter (e) --- "Western blot of his-tag P30, his-tag GRA2 and his-tag ROP2 with sero- positive serum of mice and human" --- p.98
Chapter (f) --- "Western blot of T. gondii lysate by anti-serum against his-tag GRA2, his-tag P30 and his-tag ROP2" --- p.99
Chapter 3.3.3 --- Measurement of cellular immune responses in his-tag MEA vaccinated mice --- p.99
Chapter (a) --- Isolation of spleenocytes from vaccinated mice --- p.99
Chapter (b) --- T-cell proliferation assay --- p.100
Chapter (c) --- Determination of Thl/ Th2 cytokines expression profile by RT-PCR --- p.100
Chapter 3.3.4 --- Protection efficacy of different vaccine constructs against lethal challenge --- p.102
Chapter 3.4 --- Results --- p.102
Chapter 3.4.1 --- Construction of pET-GRA2 and pET-ROP2 --- p.102
Chapter 3.4.2 --- "Expression and characterization of his-tag P30, his-tag GRA2 and his-tag ROP2" --- p.106
Chapter 3.4.3 --- Measurement of cellular immune responses in his-tag MEA vaccinated mice --- p.116
Chapter 3.4.4 --- Protection efficacy of different vaccine constructs against lethal challenge --- p.119
Chapter 3.5 --- Discussion --- p.121
Chapter 3.5.1 --- Construction of pET-GRA2 and pET-ROP2 --- p.121
Chapter 3.5.2 --- "Expression and characterization of his-tag P30, his-tag GRA2 and his-tag ROP2" --- p.121
Chapter 3.5.3 --- Measurement of cellular immune responses in his-tag MEA vaccinated mice --- p.124
Chapter 3.5.4 --- Protection efficacy of different vaccine constructs against lethal challenge --- p.126
Chapter Chapter 4 --- : Expression of the multi-epitopic antigen in Arabidopsis thaliana --- p.129
Chapter 4.1 --- INTRODUCTION --- p.129
Chapter 4.1.1 --- Transgenic plants as vaccine production systems --- p.129
Chapter (a) --- Advantages of using plants as bioreactor --- p.130
Chapter (b) --- Transgenic plants for vaccine production --- p.131
Chapter (c) --- Limitations --- p.135
Chapter (i) --- Low yields --- p.135
Chapter (ii) --- Plant-specific glycans --- p.135
Chapter 4.1.2 --- Model plant - Arabidopsis thaliana --- p.136
Chapter 4.1.3 --- Strategies for expressing the transgene (MEA) in plant --- p.137
Chapter (a) --- Seed-specific phaseolin promoter --- p.138
Chapter (b) --- Lysine-rich protein (LRP) as fusion partner --- p.138
Chapter (c) --- Fusion protein with membrane anchor for targeting to specific vacuolar compartments --- p.139
Chapter 4.2 --- MATERIALS --- p.141
Chapter 4.2.1 --- Bacterial strains --- p.141
Chapter 4.2.2 --- Arabidopsis strains --- p.141
Chapter 4.2.3 --- Chemicals --- p.141
Chapter 4.2.4 --- Antibiotics --- p.141
Chapter 4.2.5 --- Nucleic acids --- p.141
Chapter 4.2.6 --- Solutions --- p.142
Chapter 4.2.7 --- Enzymes and buffers --- p.142
Chapter 4.2.8 --- Primers --- p.143
Chapter 4.3 --- METHODS --- p.144
Chapter 4.3.1 --- Construction of plant expression vectors --- p.144
Chapter 4.3.1.1 --- Strategy 1: Lysine-rich protein (LRP) fusion --- p.144
Chapter a) --- Construction of pBI121/ Phaseolin Promoter/ LRP1/ MEA/ his-tag/ phaseolin terminator --- p.149
Chapter b) --- Construction of pBI121/ Phaseolin Promoter/ LRPIIA/ MEA/ phaseolin terminator --- p.150
Chapter 4.3.1.2 --- Strategy 2: Fusion proteins with membrane anchor for vesicle targeting to specific vesicle compartments --- p.151
Chapter ai) --- Construction of pBI121/Phaseolin Promoter/ SP/ MEA/ 491/NOS terminator --- p.156
Chapter aii) --- Construction of pBI121/Phaseolin Promoter/ SP/ MEA(J)/ 526/ NOS terminator --- p.157
Chapter 4.3.1.3 --- Construction of transgenic control pBI121/phaseolin promoter/ MEA/ phaseolin terminator --- p.157
Chapter 4.3.2 --- Agrobacterium-mediated transformation of Arabidopsis thaliana by vaccum infiltration --- p.160
Chapter (a) --- Preparation of competent cell for Agrobacterium GV3101 --- p.160
Chapter (b) --- Transformation of electro-competent agrobacterium with plant expression vector by electroporation --- p.160
Chapter (c) --- PCR screening for agrobacterium carrying the desirable plant expression vector --- p.161
Chapter (d) --- Vacuum infiltration --- p.161
Chapter 4.3.3 --- Screening of homozygous transgenic plants with single insertion of transgene --- p.162
Chapter 4.3.4 --- Detection of MEA or MEA fusion protein in transgenic plants --- p.163
Chapter (a) --- Extraction of seed protein (Soluble and membrane fractions) --- p.163
Chapter (b) --- Western blot of seed protein by anti-serum against HisMEA --- p.163
Chapter 4.4 --- RESULTS --- p.164
Chapter 4.4.1 --- Construction of plant expression vectors and transgenic A. thaliana --- p.164
Chapter (a) --- Construction of pBI121/ phaseolin promoter/ LRPI or LRP II/ MEA/ Phaseolin terminator --- p.164
Chapter (b) --- Construction of pBI121/ PP/ SP/ MEA(J)/ 491 or 526/ PT --- p.165
Chapter (c) --- Construction of transgenic control pBI121/ PP/ MEA/ PT --- p.166
Chapter (d) --- Agrobacterium-mediated transformation of A. thaliana --- p.167
Chapter 4.4.2 --- Screening of homozygous transgenic plant with single insertion of transgene --- p.172
Chapter 4.4.3 --- Molecular analysis of the MEA/ MEA fusion in transgenic plants --- p.181
Chapter 4.5 --- DISCUSSION --- p.188
Chapter 4.5.1 --- Construction of plant expression vector --- p.188
Chapter 4.5.2 --- Screening of homozygous transgenic plant with single insertion of transgene --- p.189
Chapter 4.5.3 --- Molecular analysis of transgenic MEA/ MEA fusion in transgenic plants --- p.190
Chapter Chapter 5 --- : General Discussion --- p.193
Chapter 5.1 --- Development of a vaccine for toxoplasmosis: current status --- p.193
Chapter 5.1.1 --- Current status --- p.193
Chapter (a) --- Vaccine for agricultural animals --- p.193
Chapter (b) --- Other vaccine candidates and the protective response induced --- p.194
Chapter 5.1.2 --- Appropriate vaccine design - the multi-epitopic antigen (MEA) --- p.194
Chapter 5.2 --- "Expression, Characterization and Immunological studies of the MEA expressed in prokaryotic system" --- p.195
Chapter 5.2.1 --- Expression and characterization of E. coli- expressed his-tag MEA --- p.195
Chapter 5.2.2 --- Immunological studies on the E. coli- expressed his-tag MEA --- p.196
Chapter 5.3 --- Expression of MEA in transgenic plants --- p.197
Chapter 5.4 --- Preclinical Safety Assessment: Considerations in Vaccine Development --- p.198
References --- p.200

Il seguente lavoro nasce dal proposito di approfondire le conoscenze relative alla caratterizzazione della frazione proteica e polipeptidica nella birra, allo scopo di determinare, qualitativamente e quantitativamente, sequenze peptidiche potenzialmente immunogeniche, responsabili dell’induzione della malattia celiaca. Dopo una panoramica generale, nella quale si richiamano concetti e acquisizioni sui processi di produzione della birra in considerazione della potenziale permanenza di proteine o loro derivati idrolitici, viene brevemente trattato il problema della malattia celiaca. Si passa quindi alla disamina degli studi condotti in passato e più recentemente sulla caratterizzazione delle proteine dell’orzo (ordeine) nel prodotto finito. La sezione prettamente sperimentale del lavoro di tesi è focalizzata sulla caratterizzazione delle proteine e in particolare del materiale peptidico a basse masse molecolari, in due birre commerciali italiane scelte come campioni rappresentativi. Infine, alla luce delle sequenze polipeptidiche identificate nella birra, viene analizzato e discusso il potenziale ruolo nell’induzione della malattia celiaca.

Leung Kwan-chi.
Thesis (Ph.D.)--Chinese University of Hong Kong, 1994.
Includes bibliographical references (leaves 215-223).
ACKNOWLEDGEMENTS --- p.i
ABSTRACT --- p.ii
ABBREVIATIONS --- p.iii
Chapter CHAPTER 1 --- INTRODUCTION --- p.1
Chapter 1.1 --- Brief description of Momordica charantia --- p.2
Chapter 1.2 --- Toxicity of RIPs and their potential uses in the treatment of AIDS --- p.3
Chapter 1.3 --- General mechanism of action of RIPs --- p.6
Chapter 1.4 --- Structure of αMMC --- p.7
Chapter 1.5 --- "Antigenicities of αMMC, BMMC and TCS" --- p.13
Chapter 1.6 --- "Immunosuppressive properties of the abortifacient proteins αMMC, BMMC and TCS" --- p.14
Chapter 1.7 --- Objectives of our study --- p.15
Chapter CHAPTER 2 --- EXPRESSION OF FULL-LENGTH αMMC cDNA --- p.20
Chapter 2.1 --- Expression of αMMC cDNA as a fusion protein --- p.22
Chapter 2.1.1 --- Materials and methods --- p.22
Chapter 2.1.1.1 --- Construction of fusion vector pRIT2T/MMC --- p.22
Chapter 2.1.1.2 --- Preparation of αMMC insert by PCR --- p.26
Chapter 2.1.1.3 --- Cloning of αMMC cDNA into fusion vector pRIT2T --- p.27
Chapter 2.1.1.4 --- Transformation --- p.28
Chapter 2.1.1.5 --- DNA sequencing --- p.29
Chapter 2.1.1.6 --- Expression of protein A-αMMC fusion cDNA --- p.30
Chapter 2.1.1.7 --- Preparation of fusion αMMC for affinity chromatography --- p.31
Chapter 2.1.1.8 --- Affinity chromatography of Protein A-αMMC fusion protein --- p.31
Chapter 2.1.1.9 --- Cleavage of protein A-αMMC fusion protein by factor Xa --- p.32
Chapter 2.1.1.10 --- SDS-PAGE analysis --- p.33
Chapter 2.1.1.11 --- Western blot analysis --- p.33
Chapter 2.1.1.12 --- Assay of biological activity --- p.35
Chapter 2.1.2 --- Results --- p.37
Chapter 2.1.2.1 --- Construction of pRIT2T/MMC --- p.37
Chapter 2.1.2.2 --- DNA sequencing --- p.40
Chapter 2.1.2.3 --- Expression of protein A-αMMC fusion cDNA --- p.42
Chapter 2.1.2.4 --- Purification of protein A-αMMC fusion protein --- p.45
Chapter 2.1.2.5 --- Cleavage of protein A-aMMC fusion protein --- p.49
Chapter 2.1.2.6 --- Assay of biological activity --- p.49
Chapter 2.1.3 --- Discussion --- p.51
Chapter 2.2 --- Expression of αMMC cDNA as an unfused protein --- p.52
Chapter 2.2.1 --- Materials and methods --- p.52
Chapter 2.2.1.1 --- Construction of the plasmid pET/MMC --- p.52
Chapter 2.2.1.2 --- Preparation of αMMC insert by PCR --- p.56
Chapter 2.2.1.3 --- Enzyme digestions --- p.57
Chapter 2.2.1.4 --- Ligation --- p.58
Chapter 2.2.1.5 --- Transformation --- p.59
Chapter 2.2.1.6 --- Screening for αMMC inserts --- p.59
Chapter 2.2.1.7 --- DNA sequencing --- p.60
Chapter 2.2.1.8 --- Expression of unfused aMMC cDNA --- p.60
Chapter 2.2.1.9 --- SDS-PAGE analysis --- p.61
Chapter 2.2.1.10 --- Western blot analysis --- p.62
Chapter 2.2.1.11 --- Purification of recombinant αMMC --- p.62
Chapter 2.2.1.12 --- Biological activity of recombinant αMMC --- p.63
Chapter 2.2.1.13 --- Radioimmunoassay --- p.63
Chapter 2.2.2 --- Results --- p.67
Chapter 2.2.2.1 --- Screening of pET/MMC --- p.67
Chapter 2.2.2.2 --- DNA sequencing --- p.69
Chapter 2.2.2.3 --- Expression of unfused αMMC cDNA --- p.69
Chapter 2.2.2.4 --- Radioimmunoassay --- p.72
Chapter 2.2.2.5 --- Purification of recombinant αMMC --- p.74
Chapter 2.2.2.6 --- Biological activity of recombinant αMMC --- p.74
Chapter 2.2.3 --- Discussion --- p.80
Chapter CHAPTER 3 --- EXPRESSION OF MODIFIED FORMS OF αMMC cDNA --- p.82
Chapter 3.1 --- Expression of deletion fragments of αMMC cDNA --- p.83
Chapter 3.1.1 --- Materials and methods --- p.83
Chapter 3.1.1.1. --- Construction of deletion mutants --- p.83
Chapter 3.1.1.1.1 --- Modification of pRIT2T/MMC --- p.86
Chapter 3.1.1.1.2 --- Preparation of closed circular DNA --- p.86
Chapter 3.1.1.1.3 --- Alpha-phosphorothioate nucleotide --- p.87
Chapter 3.1.1.1.4 --- Exo III digestion --- p.89
Chapter 3.1.1.1.5 --- Ligation --- p.89
Chapter 3.1.1.1.6 --- Transformation --- p.90
Chapter 3.1.1.1.7 --- Screening of deletion subclones --- p.91
Chapter 3.1.1.2 --- Confirmation of sequences --- p.91
Chapter 3.1.1.3 --- Expression of deletion mutants --- p.92
Chapter 3.1.1.4 --- Purification of deletion mutants --- p.92
Chapter 3.1.1.5 --- Cleavage of deletion mutants --- p.93
Chapter 3.1.1.6 --- Subcloning of the αMMC cDNA fragments --- p.94
Chapter 3.1.1.7 --- Expression of the unfused deletion --- p.96
Chapter 3.1.2 --- Results --- p.97
Chapter 3.1.2.1 --- Designation of the deletion mutants --- p.97
Chapter 3.1.2.2 --- Screening of deletion mutants --- p.98
Chapter 3.1.2.3 --- DNA sequencing --- p.100
Chapter 3.1.2.4 --- Expression of deletion mutants --- p.109
Chapter 3.1.2.5 --- Purification of the fusion fragments --- p.111
Chapter 3.1.2.6 --- Digestion of deletion mutants by factor Xa --- p.113
Chapter 3.1.2.7 --- Subcloning of αMMC deletion fragments --- p.115
Chapter 3.1.2.8 --- Expression of the unfused aMMC deletion --- p.117
Chapter 3.1.3 --- Discussion --- p.119
Chapter 3.2 --- Expression of a chimeric αMMC/TCS cDNA --- p.121
Chapter 3.2.1 --- Materials and methods --- p.122
Chapter 3.2.1.1 --- Construction of the MMC/TCS chimeric plasmid --- p.122
Chapter 3.2.1.1.1 --- Digestion of pfG104 - Preparation of GH1100 --- p.125
Chapter 3.2.1.1.2 --- Preparation of the GH405 fragment --- p.125
Chapter 3.2.1.1.3 --- Digestion of pACYC177 --- p.126
Chapter 3.2.1.1.4 --- "Dephosphorylation, ligation and transformation" --- p.126
Chapter 3.2.1.1.5 --- Confirmation of insert orientation --- p.127
Chapter 3.2.1.1.6 --- "Preparation of a fragment without PstI, ScaI" --- p.128
Chapter 3.2.1.1.7 --- Preparation of the 750-bp TCS fragment --- p.128
Chapter 3.2.1.1.8 --- Ligation of the TCS fragment --- p.129
Chapter 3.2.1.1.9 --- Cleavage of pACYC177/TCS with ScaI and PstI --- p.129
Chapter 3.2.1.1.10 --- Preparation of the PstI/HhaI-digested αMMC --- p.130
Chapter 3.2.1.1.11 --- Ligation of the 252-bp fragment --- p.131
Chapter 3.2.1.1.12 --- Cloning of MMC/TCS chimeric fragment --- p.131
Chapter 3.2.1.2 --- Expression of pET/MMC-TCS --- p.132
Chapter 3.2.1.3 --- SDS-PAGE analysis --- p.133
Chapter 3.2.1.4 --- Western blot analysis --- p.134
Chapter 3.2.1.5 --- Purification of MMC-TCS chimeric protein --- p.134
Chapter 3.2.2 --- Results --- p.135
Chapter 3.2.2.1 --- Construction of pET/MMC-TCS --- p.135
Chapter 3.2.2.2 --- Expression of TCS/MMC chimeric cDNA --- p.140
Chapter 3.2.2.3 --- Purification of MMC-TCS chimeric protein --- p.142
Chapter 3.2.2.4 --- Reactivity of MMC-TCS chimeric protein with various antisera --- p.145
Chapter 3.2.3 --- Discussion --- p.146
Chapter CHAPTER 4 --- SCREENING OF αMMC IMMUNO-REACTIVE FRAGMENTS FROM A RANDOM FRAGMENT LIBRARY --- p.148
Chapter 4.1 --- Materials and methods --- p.150
Chapter 4.1.1 --- Description of the pTOPE vector --- p.150
Chapter 4.1.2 --- Construction of an αMMC random fragment library --- p.152
Chapter 4.1.2.1 --- Preparation of the cDNA insert of αMMC --- p.155
Chapter 4.1.2.1.1 --- Large scale prearation of theE plasmid MMC18p8 --- p.155
Chapter 4.1.2.1.2 --- Digestion of the plasmid MMC18p8 with EcoRI --- p.156
Chapter 4.1.2.1.3 --- Electro-elution --- p.157
Chapter 4.1.2.2 --- DNase I digestion --- p.158
Chapter 4.1.2.3 --- Fractionation of DNA fragments --- p.159
Chapter 4.1.2.3.1 --- Electrophoresis --- p.159
Chapter 4.1.2.3.2 --- Electro-elution --- p.160
Chapter 4.1.2.4 --- Single dA Tailing --- p.161
Chapter 4.1.2.5 --- Ligation --- p.162
Chapter 4.1.2.6 --- Transformation --- p.162
Chapter 4.1.2.7 --- Controls --- p.163
Chapter 4.1.2.7.1 --- Full-length αMMC cDNA control --- p.163
Chapter 4.1.2.7.2 --- T-Vector ligation control --- p.164
Chapter 4.1.2.8 --- Storage of the fragment library --- p.164
Chapter 4.1.3 --- Immunoscreening of the random fragment library OF αMMC --- p.165
Chapter 4.1.3.1 --- Anti-αMMC sera --- p.165
Chapter 4.1.3.2 --- Purification of anti-αMMC sera --- p.165
Chapter 4.1.3.3 --- Colony lift --- p.167
Chapter 4.1.3.4 --- Induction of expression --- p.169
Chapter 4.1.3.5 --- Colony lysis --- p.169
Chapter 4.1.3.6 --- Immunoscreening --- p.170
Chapter 4.1.4 --- PCR screening of inserts --- p.170
Chapter 4.1.5 --- Amplification of positive signals --- p.172
Chapter 4.1.6 --- Dot blot --- p.173
Chapter 4.1.7 --- Confirmation of positive signals by Western blotting --- p.174
Chapter 4.1.8 --- Analysis of positive clones by DNA sequencing --- p.175
Chapter 4.1.9 --- Analysis of 3-dimensional structure of αMMC --- p.176
Chapter 4.1.10 --- Effect of a monoclonal anti-αMMC antibody (#A1) on ribosome-inactivating activity of aMMC --- p.176
Chapter 4.2 --- Results --- p.178
Chapter 4.2.1 --- Theoretical considerations --- p.178
Chapter 4.2.2 --- Construction of a random fragment library of αMMC cDNA --- p.180
Chapter 4.2.3 --- Screening for immuno-reactive fragments of αMMC --- p.183
Chapter 4.2.4 --- Confirmation of positive signals by Western blotting --- p.186
Chapter 4.2.5 --- Estimation of fragment sizes by PCR --- p.188
Chapter 4.2.6 --- Analysis of the fragment sequences --- p.190
Chapter 4.2.7 --- Cross-reactivity of the immuno-reactive fragments --- p.194
Chapter 4.2.8 --- Effect of a monoclonal anti-αMMC antibody (#A1) on ribosome-inactivating activity of αMMC --- p.196
Chapter 4.3 --- Discussion --- p.198
Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.200
Concluding remarks --- p.214
REFERENCES --- p.215
APPENDIXES GENERAL PROCEDURES --- p.224
Chapter A.l --- DNA sequencing --- p.224
Chapter A.2 --- Purification of DNA with Gene Clean --- p.229
Chapter A.3 --- Purification of primers after synthesis --- p.230
Chapter A.4 --- Purification of plasmid DNA by Magic Prep (Promega) --- p.232
Chapter A.5 --- Large-scale preparation of plasmid DNA by QIAGEN --- p.234
Chapter A.6 --- Lowry protein determination --- p.236
Chapter A.7 --- Preparation of acid phenol --- p.237
Chapter A.8 --- SDS-polyacrylamide gel electrophoresis --- p.238