Made available in DSpace on 2014-08-20T13:32:53Z (GMT). No. of bitstreams: 1 dissertacao_luana_alves_dummer.pdf: 909354 bytes, checksum: d2af8cc0772177718829cf7f15391d43 (MD5) Previous issue date: 2008-06-12 / Outbreaks of fatal meningoencephalitis caused by Bovine Herpesvirus type 5 (BoHV-5) cause important economic losses in national trade of bovine beef. Commercial vaccines developed against Bovine Herpesvirus type 1 can protect cattle of neurological disease caused by BoHV-5. These cross-reactions are due to its genetic and antigenic similarities of both BoHV-1 and 5. However, these vaccines can not prevent latent infection of BoHV-5, even viral spread to healthy animals. This way, new vaccines strategies are based on envelope glycoproteins and are focusing safety, economic viability and on prevention of BoHV-5 latency and spread. This glycoprotein acts in initial steps of viral infection and glycoprotein D has drawn the attention in studies carried out with BoHV-1 and other homologous herpesvirus. The gD acts on viral membrane fusion with permissive cells through glycoproteins and host receptors interactions, so it is essential for viral entry. A vaccine that is capable to stimulate specific humoral and cellular immunity against BoHV-5 must be developed. The aim of this work was the production of a recombinant truncated form of gD of BoHV-5 in yeast Pichia pastoris and its antigenicity and immunogenicity evaluation. Data show that yeast P. pastoris can express the truncated form of BoHV-5 gD to the supernatant due to its secretion of ~190mg/L of recombinant protein, simplifying protein purification. The recombinant protein was successfully recognized by antibodies of animals immunized with BoHV-5, suggesting its antigenicity and immunogenicity and that native protein characteristic are conserved. The data presented herein allow the design of future studies aiming at expression optimization and further immunogenicity evaluation, as well as its capacity to neutralize the virus in biological models and on cattle. / Surtos de meningoencefalites fatais causadas pelo Herpesvírus Bovino tipo 5 (BoHV-5) ocasionam importantes perdas econômicas no comércio nacional de carne bovina. Vacinas comerciais destinadas ao Herpesvírus Bovino tipo 1 são capazes de impedir o aparecimento de sintomatologia clínica do BoHV-5. Estas reações cruzadas se devem a existência de semelhanças genéticas e antigênicas existentes entre ambos os vírus. No entanto, estas vacinas não impedem o estabelecimento da infecção latente e a disseminação do BoHV-5 para animais não imunizados. Desta forma, estratégias que visem à produção de vacinas seguras, economicamente viáveis e que possam ser capazes de impedir a latência do BoHV-5 e sua disseminação estão focadas para glicoproteínas localizadas no envelope viral e que atuam nas etapas iniciais da infecção. Dentre estas, a glicoproteína D tem se destacado em estudos realizados com BoHV-1 e outros herpesvírus homólogos. A gD atua na fusão do envelope viral com a membrana da célula permissiva no hospedeiro através de interações com receptores celulares e com outras glicoproteínas virais, sendo essencial para a entrada do capsídeo na célula. Assim, uma vacina que seja capaz de estimular respostas humorais e celulares contra esta glicoproteína deve ser desenvolvida. Os objetivos deste trabalho foram à produção da forma truncada da gD do BoHV-5 em levedura Pichia pastoris e a avaliação desta quanto a sua antigenicidade e imunogenicidade. Os resultados demonstram que a Pichia pastoris expressou a forma truncada da gD de BoHV-5 secretando para o meio de cultivo ~190mg/L da proteína recombinante, facilitando a purificação da mesma. Testada quanto a sua a sua antigenicidade e imunogenicidade, a proteína recombinante foi reconhecida por anticorpos de animais imunizados com o BoHV-5, sugerindo que características da proteína nativa foram conservadas na proteína recombinante. Os dados apresentados irão permitir o desenvolvimento de estudos 6 futuros com o objetivo de aperfeiçoar a expressão da proteína recombinante e avaliar a sua capacidade de neutralizar o vírus na espécie-alvo.

Made available in DSpace on 2014-08-20T13:32:57Z (GMT). No. of bitstreams: 1 tese_lorena_leonardo_souza.pdf: 798251 bytes, checksum: b0213429356f32517eb7b25ac76b2eeb (MD5) Previous issue date: 2009-12-17 / The rabies virus has two major antigens: the nucleoprotein, a conserved internal protein antigenically and genetically and glycoprotein, a protein responsible for the external adsorption of virus two the host cell and induction of neutralizing antibodies. The development of recombinant DNA technology has opened a new perspective on the control of rabies, since recombinant vaccines have residual pathogenicity and are produced with the antigenic proteins of the virus, without their presence. Furthermore, recombinant proteins can be expressed in order to be used in diagnosis. The objective of this study was to review the literature about rabies, cloning and express the nucleoprotein and glycoprotein of rabies virus using the system Pichia pastoris and evaluate the antigenicity and the immunogenicity of these proteins by Dot blotting, SDS page, Western blotting and ELISA. Glycoprotein synthetic antigen proved to be recognized by anti-rabies from animals experimentally infected with rabies virus strain CVS. And recombinant nucleoprotein expression was confirmed by the techniques of Dot blotting and Western blotting to be recognized by monoclonal anti-histidine. Thus, we conclude that the cloning and expression of synthetic glycoprotein and cloinig and expression nucleoprotein rabies virus by the yeast P. pastoris has been effective, which makes these products an alternative for the production of immunobiological. / O vírus da raiva apresenta dois antígenos principais: a nucleoproteína, uma proteína interna conservada antigênica e geneticamente e a glicoproteína, uma proteína externa responsável pela adsorção do vírus à célula hospedeira e pela indução da produção de anticorpos neutralizantes. O desenvolvimento da tecnologia do DNA recombinante iniciou uma nova perspectiva no controle da Raiva, já que vacinas recombinantes não têm patogenicidade residual e são produzidas com as proteínas antigênicas do vírus, sem sua presença. Além disso, as proteínas recombinantes podem ser expressas com a finalidade de serem usadas em diagnóstico. O objetivo deste trabalho foi clonar e expressar a glicoproteína e a nucleoproteína do vírus da raiva utilizando o sistema Pichia pastoris e avaliar a antigenicidade e imunogenicidade destas proteínas através do Dot blotting, SDS page, Western blotting, inibição da imunofluorescência e ELISA. A glicoproteína demonstrou ser antigênica ao ser reconhecida por anticorpos anti-rábicos provenientes de animais experimentalmente infectados com o vírus rábico cepa CVS. A nucleoproteína recombinante teve sua expressão confirmada pelas técnicas de Dot blotting e Western blotting ao ser reconhecida por anticorpos monoclonais anti-histidina. Podemos concluir que a levedura P. pastoris é um sistema eficiente para clonagem e expressão da nucleoproteína e glicoproteína do vírus rábico.

Le virus de la variole ovine est omniprésent dans les élevages de petits ruminants dans les pays d’Afrique du Nord et particulièrement en Tunisie malgré les campagnes de vaccination annuelles mises en place par les autorités vétérinaires du pays. L’optimisation de la souche vaccinale utilisée passe par le développement de vaccins dits de nouvelle génération tels que les vaccins sous unitaires utilisant des protéines reconnues pour induire une réponse humorale protectrice chez l’animal immunisé. Ceci pourrait être une alternative aux stratégies de lutte actuelles permettant de limiter la dissémination du virus en Tunisie. Peu de données existent sur les antigènes protecteurs spécifiques des virus du genre Capripoxvirus. Ce travail de thèse a ciblé, par homologie aux protéines du virus de la vaccine, quatre protéines du genre Capripoxvirus appartenant au virus de la dermatose nodulaire contagieuse potentiellement immuno-dominantes nommées LSDV60, LSDV117, LSDV122 etLSDV141 respectivement homologues des protéines L1, A27, A33 et B5. En premier lieu, une analyse structurale in silico a permis d’identifier les domaines essentiels de chaque protéine et de vérifier le taux de conservation de ces protéines parmi différents virus appartenant à la famille des poxvirus. Une analyse structurale approfondie mettant en évidence la structure primaire, secondaire et tertiaire de la protéine A27 a été réalisée. Suite à cette étude structurale, les protéines ont été produites dans deux systèmes d’expression différents ; le système eucaryote et le système baculovirus-cellules d’insectes afin de caractériser leur antigénicité vis-à-vis de sérums provenant d’animaux immunisés ou éprouvés.La reconnaissance des protéines d’intérêt en vecteur d’expression eucaryote n’a pas été concluante. En revanche, le système d’expression BEVS a permis la production de la protéine A27 (L1, A33 et B5 encours) avec succès sous forme soluble qui a été correctement reconnue par des sérums provenant de caprins naïfs challengés. La mise en évidence de formes trimériques et hexamériques confirment sonantigénicité. Une immunodétection des peptides correspondants à la protéine A27 synthétisés surmembranes (PepScan) combinée à une analyse in silico ont permis d'identifier des zones susceptibles de constituer des régions épitopiques reconnues situés majoritairement en partie N terminale de la protéine. / The sheep pox virus is omnipresent in small ruminant farms in North African countries andparticularly in Tunisia despite the annual vaccination campaigns set up by the Tunisian veterinaryauthorities. The optimization of the used vaccine strain involves the development of the so-called newgeneration vaccines such as subunit vaccines and this, using proteins recognized to induce a protectivehumoral response in the immunized animal. This could be considered as an alternative to currentcontrol strategies limiting virus spread in Tunisia. Few data exist on protective antigens specific toviruses in the genus Capripoxvirus. By homology to vaccinia virus proteins, this thesis work hastargeted four proteins in the genus Capripoxvirus belonging to the potentially immuno-dominantcontagious nodular dermatosis virus named LSDV60, LSDV117, LSDV122 and LSDV141respectively homologues of proteins L1, A27, A33 and B5. First, an in silico structural analysis hasallowed to identify the essential domains of each protein and to check the conservation rate of theseproteins among different viruses belonging to the poxvirus family. A thorough structural analysisidentifying the primary, secondary and tertiary structure of the A27 protein was conducted. Followingthis structural study, the proteins were produced in two different expression systems, namely theeukaryotic system and the baculovirus-insect cell system, in order to characterize their antigenicity tosera from immunized or proven animals. The recognition of the proteins of interest in the eukaryoticexpression vector has not been conclusive. On the other hand, the BEVS expression systemsuccessfully allowed the production of the A27 protein (L1, A33 and B5 in progress) in a solubleform, which was correctly recognized by sera from challenged naïve goats. Identifying trimeric andhexameric forms confirms its antigenicity. An immunodetection of the peptides corresponding toprotein A27 synthesized on membranes (PepScan) combined with an in silico analysis led to identifyzones capable of constituting recognized epitopic regions located predominantly in part N-terminal ofthe protein.

Abstract Potential lethal diarrhoea caused by enterotoxigenic Escherichia coli strains is one of the most common diseases in young pigs. It can be cured by single-chain antibody fragments (scFv), which can be produced in recombinant microorganisms. Pichia pastoris, a methylotrophic yeast, is generally considered an interesting production system candidate, as it can secrete properly folded proteins. These proteins accumulate in high concentrations during fermentation, reducing the cost for product recovery. Strong inducible AOX1 promoter, widely used in P. pastoris for fast, inexpensive production, is typically induced by methanol. The high oxygen demand of methanol metabolism makes oxygen supply a major parameter in cultivations requiring special process design strategies. In standard fed-batch cultivation, dissolved oxygen concentration inside a bioreactor is kept at a certain level by pumping air and pure oxygen into the reactor. There are safety concerns over the handling of oxygen, especially at a large scale. Therefore, there is a need to develop a production process under oxygen-limited conditions. This dissertation studies the development of a cost-efficient production process of scFv in P. pastoris. Both methanol and oxygen parameters influence the production process and the objective was to find a robust production process. Fed-batch cultivations were performed in a 10 L scale bioreactor. The effects of lower oxygen level, methanol concentration, glycerol feeding duration and specific substrate-uptake rates on product formation were studied. A P. pastoris GS115 his4 strain under an AOX1 promoter system expressing scFv was used in this study. The fed-batch fermentations were carried out in a bioreactor with basal salt media. In this doctoral dissertation, a process was developed for a single-chain antibody fragment (scFv) production in P. pastoris. The product levels of 3.5 g L-1 scFv in culture supernatant were achieved and a production process was designed without additional need of pure oxygen, thus relieving safety requirements and lowering the amount of methanol. The process developed during this research may potentially be utilised by both academia and industry having interests in expressing proteins in P. pastoris. The methanol-uptake control strategy is beneficial for those products that suffer from degradation or modification during limited feeding of methanol. / Tiivistelmä Enterotoksigeenisten E.coli kantojen aiheuttama ripuli on porsaiden tavallisimpia tauteja, joka voi johtaa jopa kuolemaan. Tautia voidaan hoitaa yhdistelmä-DNA-tekniikalla tuotetuilla vasta-ainefragmenteilla (scFv). Metylotrofista Pichia pastoris hiivaa pidetään kiinnostavana vasta-ainefragmenttien tuottoisäntänä, koska se pystyy erittämään oikealla tavalla laskostuneita proteiineja. Näitä proteiineja kertyy fermentointiprosessissa solujen ulkopuolelle korkeina pitoisuuksina, mikä vähentää tuotteiden talteenottokustannuksia. Vahva metanolilla indusoituva AOX1-promoottori on laajassa käytössä P. pastoris tuottosysteemissä tuoton nopeuden ja alhaisten kustannusten ansiosta. Metanolin aineenvaihdunta vaatii paljon happea, joten riittävän tehokas hapen liuottaminen on tärkeimpiä fermentointiparametreja ja vaatii erityisiä prosessin toteutusstrategioita. Perinteisessä fed-batch-fermentoinnissa liuenneen hapen pitoisuus bioreaktorissa pidetään halutulla tasolla lisäämällä ilmaa ja puhdasta happea reaktoriin. Koska hapen käsittelyyn liittyy turvallisuusriskejä erityisesti teollisuusmittakaavassa, happirajoitteisissa olosuhteissa toimiva tuotantoprosessi olisi hyödyllinen. Tässä väitöstutkimuksessa kehitettiin kustannustehokasta prosessia scFv-:n tuottoon P. pastoris hiivalla. Metanoliin ja happeen liittyvät parametrit ovat olennaisia prosessiin vaikuttavia tekijöitä. Tavoite oli kehittää yksinkertainen ja käytännöllinen prosessi. Työssä tutkittiin alhaisen happitason, metanolin pitoisuuden, glyserolisyötön keston ja substraattien spesifisten kulutusnopeuksien vaikutuksia tuotteen muodostumiseen 10 litran bioreaktorissa. Isäntäkantana oli P. pastoris GS115 his4, jossa scFv-ekspressiota säädeltiin AOX1 promoottorilla. Fed-batch fermentointien kasvatusalustana käytettiin Basal Salt Medium alustaa (BSM). Väitöstyössä kehitettiin tavoitteiden mukainen vasta-ainefragmenttien tuottoprosessi P.pastoris hiivalle. Menetelmällä saavutettiin tuotepitoisuus 3,5 g L-1 kasvatusliemen supernatantissa ilman puhtaan hapen lisäystarvetta, ja siten metanolin kulutus väheni ja prosessiturvallisuus parani verrattuna perinteisiin prosesseihin. Kehitetty prosessi soveltuu käytettäväksi sekä akateemisessa tutkimuksessa että teollisuudessa tuotettaessa erilaisia proteiineja P. pastoris hiivalla. Metanolin kulutuksen säätöstrategia on erityisen hyödyllinen tuotteille, joilla ongelmana on proteolyysi tai muokkautuminen metanolirajoitteisessa fermentoinnissa.

Drug resistant strains of Clostridium difficile are a major health concern with over 3 million cases costing over 1 billion $ per year in the United-States. The diseases associated with these bacteria (CDAD) are toxin-mediated which offers a mean of treating and lessening the severity of CDAD symptoms. Toxin inactivation via antibodies therapy can drastically reduce CDAD morbidity and this project was aiming at investigating the large-scale production and recovery of a novel llama single domain antibody (pSJF2H-ToxA5.1) in recombinant Escherichia coli TG1 targeting C. difficile enterotoxin A (TcdA). In order to achieve these objectives, the project was divided into four segments: 1) ToxA5.1 being an intracellular recombinant protein, obtaining a high biomass production was the first step towards large-scale production. To achieve HCDC, effects of initial glucose concentration and pH-stat feeding strategy were studied; 2) Upon achieving HCDC, effects of parameters such as temperature, induction timing and media supplementation with complex nitrogen sources were investigated; 3) Once large-scale production of ToxA5.1 was obtained, the recombinant protein needed to be recovered and a selective cell lysis scheme where synergistic lysis effects of Triton X-100 and temperature were studied. And finally 4) Single-step purification using nickel nanoparticles (NNP) synthesized via a modified polyol method was studied. Combining the HCDC strategy with a temperature shift and yeast extract addition at the time of induction, ToxA5.1 concentration of 127 mg/L was obtained. Synergistic and selective cell lysis using Triton X-100 and temperature was achieved where 95% of the available ToxA5.1 was recovered and still functional while ToxA5.1 fraction in the resulting lysate increased to 27% in the cell lysate. Single-step purification was achieved using the synthesized NNP which proved to be highly selective and could be used up to five times. Diameter of the NNP synthesized was controlled by using various concentration of ranging from 131 ± 80 nm to 47 ± 20 nm. Using experimental data from binding isotherm, the ToxA5.1-NNP system was modeled.

Les besoins en protéines recombinantes dans les diverses activités des bio-industries a considérablement augmenté ces dernières années. Cependant, les procédés de leur production sont encore limités par le manque de marqueurs permettant de suivre l'expression et la purification des protéines d'intérêt, la formation des corps d'inclusion et les faibles degrés de pureté.Afin de pallier à ces difficultés, nous avons développé et mis en œuvre un nouveau procédé de production et de purification de protéines recombinantes chez Escherichia coli. Ce procédé est basé sur l'utilisation d'une cassette d'expression appelée Multitags et sur le clivage par une TEV protéase immobilisée sur une matrice de streptavidine. Le Multitags comporte, à partir de son extrémité N-terminale, un double tag d'affinité (10xHis et SBP), le domaine de fixation de l'hème du cytochrome b5 et le site de clivage de la TEV protéase. En utilisant deux modèles différents de protéine d'intérêt (MyRIP et la Pfu DNA polymérase), nous avons montré l'efficacité du cytochrome b5 dans le suivi visuel et quantitatif par mesure d'absorbance des différentes étapes de production et de purification. Nous avons obtenu plus de 90% de chacune des deux protéines de fusion dans la phase soluble. L'application d'une chromatographie double via les deux tags d'affinité 10xHis et SBP a permis d'atteindre un degré de pureté du Multitags-MyRIP et du Multitag-Pfu de 99%. Nous avons construit des colonnes protéolytiques en produisant la TEV protéase sauvage et sa version mutée (S219V) en fusion avec le Streptag II et en immobilisant ces enzymes par affinité sur colonne de streptavidine-agarose. La caractérisation des colonnes protéolytiques et leur application aux protéines recombinantes d'intérêt modèles ont montré l'avantage de cette méthode d'immobilisation en termes d'activité protéase retenue, de stabilité des enzymes, de leur réutilisation et de simplification du schéma de purification et de récupération des protéines d'intérêt à haut degré de pureté. En conclusion, ces travaux de thèse ont permis de développer et de valider des outils innovants pour l'expression et la purification de protéines recombinantes. / In recent years, the need for recombinant proteins has substantially increased in various bio-industry activities. However, actual recombinant processes are still limited by the lack of markers allowing real-time expression and purification monitoring of target proteins, by inclusion bodies formation and by low quality of purity of the products. To overcome these difficulties, we have developed a new process for production and purification of recombinant proteins in Escherichia coli. The method combines the use of a multifunctional expression cassette, termed Multitags and an immobilized modified TEV protease on a streptavidin matrix. The Multitags contains, its N-terminus, two affinity purification tags (10xHis and SBP) and as a marker tag, the heme-binding domain of cytochrome b5 followed by the TEV cleavage site. Using two model proteins (MyRIP and Pfu DNA polymerase), we have demonstrated the visual and the quantitative monitoring capability of the cytochrome b5 during the expression and purification steps. When expressed in E. coli KRX more than 90% of both fusion proteins were produced in a soluble form. In addition, high purity (99%) of Multitags-MyRIP and Multitags-Pfu was achieved after two consecutive affinity purification steps using the dual affinity tag. We also produced the wild-type and the S219V mutant TEV proteases fused to the Streptag II affinity sequence and realized their affinity immobilization on a streptavidin-agarose matrix. The characterization of the proteolytic columns and their application to the recombinant model proteins demonstrated the advantage of this immobilization method in terms of retaining activities, enzyme stabilities, possibility of reuse and simplification of the cleavage downstream steps.In conclusion, this study allowed the development and the validation of innovative tools for expression and purification of recombinant proteins.

O HIV-1 é o agente etiológico da síndrome da imunodeficiência adquirida (AIDS) e o HTLV-I da leucemia/linfoma de célula T no adulto (ATL) e da paraparesia espástica tropical ou mielopatia associada ao HTLV (HAM/TSP), principalmente. Ambos são retrovírus com genoma RNA e possuem o gene gag que codifica as proteínas p24 (HIV-1 e HTLV-1) e p19 (HTLV-1) que formam o capsídeo e a matriz do vírus, respectivamente, e o gene env que codifica as proteínas gp41 e gp120 (HIV-1) e gp21 e gp46 (HTLV-1) que compõem o envelope viral. Os primeiros anticorpos produzidos nas infecções por ambos os vírus são destinados a essas proteínas e os diferentes testes diagnósticos disponíveis no mercado usam uma combinação dessas proteínas virais. O diagnóstico precoce é de extrema importância para o controle da epidemia, tratamento dos indivíduos e planejamento dos gastos com saúde pública. Os kits diagnósticos usados em laboratórios clínicos, bancos de sangue e hospitais brasileiros para o diagnóstico destas viroses são na sua maioria de empresas estrangeiras e o Brasil despende milhares de reais importando esses materiais. No Brasil, há a necessidade e incentivo para a produção de sistemas de diagnóstico com tecnologia nacional. Neste trabalho, os genes das proteínas p24, gp41 e gp120 do HIV-1 e p19 do HTLV-1 foram clonados com sucesso em diferentes vetores e em diferentes linhagens de E. coli, porém essas proteínas não foram expressas. As proteínas gp21, p24 e gp46 do HTLV-1 foram produzidas em bactérias BL21(DE3) com vetor pET28a(+). Essas três proteínas foram solubilizadas dos corpos de inclusão, purificadas por IMAC e identificadas pelas técnicas de Western Blotting e por espectrometria de massas. As proteínas recombinantes gp21, p24 e gp46 foram reconhecidas pelos soros de indivíduos com HTLV-1 e não foram reconhecidas por soros de indivíduos com HIV-1 e saudáveis, o que confere a elas especificidade e grande potencial diagnóstico. Os resultados deste trabalho são os primeiros passos para atingir o objetivo maior de produzir todas as sete proteínas em maior escala e, por fim, chegar a produção de um kit diagnóstico sensível, específico e barato com tecnologia nacional, diminuindo os gastos com a importação destes produtos e fomentando a indústria biotecnológica nacional. / HIV-1 is the etiologic agent of acquired immunodeficiency syndrome (AIDS) and HTLV-I is the cause of adult T-cell leukemia/lymphoma (ATL) and HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). Both are retroviruses with RNA genome and possess the gene gag and env. The gag gene encodes for p24 protein (HTLV-1 and HIV-1) and p19 (HTLV-1) forming the viral capsid and matrix, respectively, and the env gene encodes for proteins gp120 and gp41 (HIV-1) and gp21 and gp46 (HTLV-1) making the viral envelope. The first antibodies produced in infections by both viruses are against these proteins and the various diagnostic tests on the market use a combination of those viral proteins. Early diagnosis is extremely important to control the epidemia, treatment of individuals and planning of public health expenditures. The diagnostic kits used in clinical laboratories, blood banks and in Brazilian hospitals for the diagnosis of these viruses are mostly from foreign companies. Brazil spends thousands of reais importing these materials. In Brazil, there is a need and incentive for the production of diagnostic systems with national technology. In this study, the genes of p24, gp41 and gp120 of HIV-1 and p19 of HTLV-1 have been successfully cloned in different vectors and different strains of E. coli, but these proteins were not expressed. The proteins gp21, gp46 and p24 of HTLV-1 were produced in bacteria BL21 (DE3) with vector pET28a (+). These three proteins were solubilized from inclusion bodies, purified by IMAC and identified by Western blotting techniques and mass spectrometry. The recombinant proteins gp21, p24 and gp46 were recognized by sera from patients with HTLV-1 and were not recognized by sera from individuals with HIV-1 and healthy people, which gives them great specificity and diagnostic potential. These results are the first steps to achieve the ultimate goal of producing all seven proteins on a larger scale and finally get the production of a diagnostic kit sensitive, specific and cheap with national technology, reducing spending on imports of these products and fostering the national biotechnology industry.

ATPDases ou ATP-difosfohidrolases são enzimas que clivam o ATP e o ADP a AMP e Pi e estão envolvidos em inibição da agregação plaquetária. No parasita Schistosoma mansoni nosso grupo identificou e clonou o gene da ATPDase1, e a proteína foi localizada na superfície do tegumento. Recentemente, clonamos o gene da ATPDase2 usando a informação do banco de dados de ESTs de S. mansoni e imunolocalizamos a sua proteína também no tegumento. ATPDase2 foi encontrada em ambas as membranas do tegumento basal e apical juntamente com a ATPDase1, entretanto ATPDase2 somente foi encontrada no espaço sincicial do tegumento. A presença de ambas as enzimas sobre a superfície externa do tegumento sugere um maior papel sobre a regulação de abundância de nucleotídeos. Análise da expressão de ambos os genes foram realizadas por RT- PCR em tempo real usando RNA de ovos, miracídeo, cercária, esquistossômulo e verme adulto. Os resultados mostraram que o gene da ATPDase1 foi mais expresso em ovos (7 vezes), adulto (6 vezes), cercária (3,5 vezes) e esquistossômulo (1,5 vezes) quando comparado ao miracídio, que foi tomado como referência. O gene da ATPDase2 foi mais expresso em ovos (16 vezes), cercária (11 vezes), miracídio (7 vezes) e verme adulto (2 vezes) quando comparado a esquistossômulo, mostrando que ambos os genes são modulados ao longo de seus estágios de ciclo de vida . Para maior caracterização destas enzimas, elas foram expressas heterologamente na levedura Pichia pastoris como proteínas de fusão com cauda de 6 histidinas e as proteínas recombinantes foram purificadas por cromatografia de afinidade com resina de Ni-NTA. As ATPDases recombinantes foram obtidas de forma ativa e medições de atividade enzimática foram realizadas. ATPDase1 - mostrou atividades ATPásica e ADPásica em torno de 650 e 160 nmoles Pi.min -1. mg-1 , respectivamente. ATPDase2 teve atividades ATPásica e ADPásica na faixa de 1050 e 250 nmoles Pi.min-1.mg-1 , respectivamente. Adicionalmente, atividades UTPásica e UDPásica também foram encontradas nestas enzimas. Estudos de dicroísmo circular com estas duas enzimas elucidaram suas estruturas secundárias. Com isto, ATPDase1 (S66 to Q507 ) teve alfa-hélice (7 %), folha-beta (45 %) e estrutura randômica (48 %), e a ATPDase2 (N83 a K564 ) mostrou conter alfa-hélice (14 %), folha-beta (33 %) e estrutura randômica (53 %). Nós mostramos que a ATPDase2 é secretada pelo parasita no meio, de forma similar como descrito para as ATP-difosfohidrolases humanas CD39L2 e CD39L4. Adicionalmente, em ensaios de inibição de penetração (cercária em camundongo) usando anticorpo anti- ATPDase1 foi mostrada uma redução em 20 % da capacidade de penetração através da pele das cercárias previamente incubadas com o anti-soro. Devido a que a expressão do gene da ATPDase2 estava mais alta em miracídio e cercária, estágios que infectam caramujos e humanos, respectivamente, postulamos que ATPDase2 poderia ajudar no processo de invasão do parasita. No estágio ovo ambos os genes estão altamente expressados sugerindo um possível envolvimento das ATPDases na resposta de proteção contra o sistema imune humano. Ensaios de proteção contra S. mansoni em camundongos, usando as ATPDases 1 e 2 como antígenos, resultaram em uma baixa proteção obtendo-se não mais que 20% na redução da carga parasitária. / ATPDases or ATP-diphosphohydrolases are enzymes that cleave ATP and ADP to AMP and Pi and are involved in inhibition of platelet aggregation. In the parasite Schistosoma mansoni our group had identified and cloned the ATPDase1 gene and localized the protein on the tegument surface. Recently, we cloned the ATPDase2 gene using S. mansoni EST databank information and we immunolocalized it also in the tegument. ATPDase 2 was found on both the apical and basal tegument membranes together with ATPDase1, but only ATPDse2 was found in the syncytium space of the tegument. The presence of both enzymes on the tegumental outer surface suggests a major role in regulation of nucleotides abundance. Expression analysis of both genes was performed by Real Time RT- PCR using RNA from eggs, miracidia, cercariae, schistosomula and adult worms. The results showed that ATPDase1 gene was more expressed in eggs (7-fold), adults (6-fold), cercariae (3.5-fold) and schistosomula (1.5-fold) when compared to miracidia, which was taken as the reference. ATPDase2 gene was more expressed in eggs (16-fold), cercariae (11-fold), miracidia (7-fold) and adult worms (2-fold) when compared to schistosomula, showing that both genes are modulated along the life cycle stages. For further characterization of these enzymes, they were expressed heterologously in the yeast Pichia pastoris as fusion proteins with hexa-histidine tags and the recombinant proteins were purified by Ni-NTA affinity chromatography. The recombinant ATPDases were obtained in active form and activity measurements were performed. ATPDase1 did show ATPase and ADPase activities about 650 and 160 nmoles Pi.min-1.mg-1 , respectively. ATPDase2 had ATPase and ADPase activities in the range of 1050 and 250 nmoles Pi.min-1.mg-1 , respectively. These results were obtained in the presence of calcium as cofactor. Additionally, UTPase and UDPase activities were found for both enzymes. Circular dichroism studies with these enzymes elucidated their secondary structures; ATPDase1 (S66 to Q507 ) has alpha helix (7%), beta sheet (45%) and random coil (48%), whereas ATPDase2 (N83 a K564 ) showed alpha helix (14%), beta sheet (33%) and random coil (53%). We found that ATPDase2 was secreted by the parasite to the medium, similar to what has been described for human CD39-L2 and CD39-L4 ATP-diphosphohydrolases. Additionally, a penetration assay (cercaria to mice) using antibody anti-ATPDase1 did show a decrease of 20% in the penetration capacity through mice skin of cercaria previously incubated with this antiserum. Because ATPDase2 gene expression was increased in miracidia and cercariae, the stages that infect snail and human, respectively, we postulate that ATPDase2 may help the parasite\'s invasion process. In the egg stage both genes were highly expressed suggesting a possible involvement of the ATPDases in the protection response of eggs against the human immune system. Assays of protection against S. mansoni in mice, using recombinant ATPDase1 and 2 as antigens, resulted in low protection obtaining no more than 20% of parasite burden reduction.

A lagarta L. obliqua tem se destacado por apresentar em sua hemolinfa proteínas com atividade biológica demonstrada em cultivos celulares. A literatura disponível não apresenta trabalhos sobre a expressão da proteína antiapoptótica de L. obliqua, então este estudo objetiva a clonagem e expressão da proteína antiapoptótica presente na hemolinfa de L. obliqua, em sistema bacteriano Escherichia coli. O RNAm extraído do tegumento de L. obliqua deu origem a um cDNA obtido por RT-PCR, o qual foi clonado em vetor pCR II-TOPO para posterior transformação de bactérias E. coli JMQC. Na expressão heteróloga, o fragmento foi subclonado em vetor pET28a e transformadas bactérias E. coli BLQC. A indução de expressão foi realizada com IPTG 1 mM. A APLOrEC purificada por cromatografia foi identificada por Western Blot. A atividade biológica da APLOrE foi analisada em células VERO e L929 após indução de morte e verificou-se que esta protegeu os cultivos induzidos com 4mM de H2O2 portanto, eficaz na manutenção estrutural do citoesqueleto destas células. / The caterpillar L. obliqua has become known for performing in their hemolymph proteins with biological activity demonstrated in cell cultures. The available literature does not provide studies on the expression of antiapoptotic protein L. oblique, so this study aims cloning and expression of anti-apoptotic protein present in the hemolymph of L. oblique, bacterial system in Escherichia coli. The extracted mRNA L. obliqua husk gave a cDNA obtained by RT-PCR, which was cloned into pCR II-TOPO vector for subsequent transformation of E. coli bacteria JMQC. The heterologous expression, the fragment was subcloned into pET28a vector and transformed E. coli bacteria BLQC. The induction of expression was performed with 1 mM IPTG. The APLOrEC purified by chromatography was identified by Western blot. The biological activity was examined in APLOrE VERO and L929 cells after death induction, and it was found that this protected crops H2O2 induced with 4mM therefore effective in the structural maintenance of the cytoskeleton of such cells.

Streptococcus pneumoniae é o patógeno causador de diversas doenças com alta mortalidade e morbidade, como meningite e pneumonia. As vacinas disponíveis baseiam-se na resposta contra o polissacarídeo capsular (PS), porém possuem elevado custo e cobertura limitada aos sorotipos vacinais. O objetivo deste estudo foi avaliar a eficácia da imunização nasal com PspAs recombinantes de família 1 (rPspA1 e rPspA2) e de família 2 (rPspA3, rPspA4 e rPspA5) em um modelo de cocolonização da nasofaringe de camundongos, utilizando isolados que expressam diferentes PspAs (PspA1 ao PspA4). Esse modelo visa analisar a eficácia da vacinação frente à exposição a diferentes pneumococos, uma situação comum, especialmente em crianças. Os experimentos deste projeto avaliaram a colonização com misturas de isolados dos sorotipos 6B e 23F e expressando PspA1, PspA2, PspA3 ou PspA4, mostrando uma análise ampla da cobertura vacinal contra pneumococo das diferentes formulações contendo as variantes do antígeno PspA e a importância desse antígeno para o desenvolvimento de uma nova vacina. / Streptococcus pneumoniae is the cause of several diseases with high mortality and morbidity, such as meningitis and pneumonia. The available vaccines are based on the response against the capsular polysaccharide (PS), but they have a high cost and coverage restricted to the vaccine serotypes. The purpose of this study was to evaluate the efficacy of nasal immunization with recombinant PspAs from family 1 (rPspA1 and rPspA2) and from family 2 (rPspA3, rPspA4 and rPspA5) in a model of co-colonization of the mouse nasopharynx, using isolates expressing different PspAs (PspA1 to PspA4). This model aims to analyze the effectiveness of vaccination upon exposure to different pneumococci, a common situation, especially in children. The experiments in this project assessed colonization with mixtures of isolates of serotypes 6B and 23F, expressing PspA1, PspA2, PspA3 or PspA4, showing a wide vaccination coverage analysis of different formulations containing the PspA variants against pneumococcus and the importance of this antigen to the development of a new vaccine.