Dissertations / Theses on the topic 'Proteine s1'

L'endoribonuclease regb du bacteriophage t4 coupe au milieu des motifs ggag des arnm. Regb purifiee est tres peu active in vitro. Son activite est stimulee environ 100 fois par la proteine ribosomique s1. In vivo, s1 est responsable de l'inactivation fonctionnelle et chimique des arnm precoces de t4 et egalement de la maturation des longs arnm precoces de t4. In vivo, tous les motifs ggag ne sont pas coupes par regb. Dans cette these, je montre que la sensibilite et la resistance a regb observees apres une infection par t4 peuvent etre reproduites in vivo dans les cellules d'e. Coli non-infectees et in vitro. Cela suggere que les motifs ggag resistants a regb apres l'infection ne sont pas des substrats, quelque soit la periode au cours du cycle ou les arnm sont faits. In vitro, a hautes concentrations, regb seule est capable de couper specifiquement les motifs ggag. L'efficacite de clivage est deja meilleure pour un bon substrat que pour un mauvais substrat. Regb possede donc l'activite enzymatique et discriminatoire. S1 stimule l'activite de regb avec les deux types d'arnm du meme ordre de grandeur. Un mauvais substrat peut etre bien coupe par regb en presence de hautes concentrations de s1, cependant, a une concentration de regb donnee, l'efficacite de coupure par regb dans les mauvais substrats atteint un plateau plus bas que pour les bons substrats. L'analyse des structures secondaires de ces deux types d'arn par sondes chmiques montre que les mauvais substrats ont des structures plus complexes autour du motif ggag que les bons substrats. Regb serait inhibee par ces structures. Dans un petit arn de 30 nucleotides, l'efficacite de coupure par regb dans un motif ggag situe dans une region simple brin est meilleure que celle localisee dans un motif structure (structure predite par calcul). En plus, s1 n'est pas necessaire pour la coupure dans ce motif ggag libre. S1 serait un arn chaperon, modifiant la structure du motif ggag de maniere a ce qu'il soit coupe.

Nous avons prepare des anticorps monoclonaux diriges contre des proteines de la sous-unite 30s du ribosome d'e. Coli en vue de leur utilisation comme sondes fonctionnelles et structurales. Nous presentons d'abord une technique de fractionnement des proteines de la sous -unite 30s du ribosome d'e. Coli par chromatographie en phase inverse sur un systeme fplc. Puis, nous decrivons l'obtention et la caraterisation d'anticorps monoclonaux diriges contre quelques proteines de la sous-unite 30s: les proteines s1, s4, s12 et s17. Ces anticorps ont permis de montrer que des modifications des conditions ioniques dans lesquelles se trouvent la sous-unite 30s affectent l'accessibilite ou la conformation d'epitopes situes a la surface des proteines s4 (ou s17) et s12. Nous avons pu mettre en evidence des changements conformationnels discrets entre deux etats differents de la sous-unite 30s. Enfin, nous presentons l'obtention et la caracterisation d'anticorps monoclonaux diriges contre la proteine s1

Le genome chloroplastique d'epinard est constitue d'une molecule d'adn circulaire (140 kbp) organisee en 4 regions: une sequence unique (lsc) et une petite sequence unique (ssc) separees par deux regions inversees repetees (ira et irb). L'expression des genes rp12, rps19 et rps19' est etudie. Les techniques de clonage et de cartographie a la nuclease s1 ont peris de montrer que le gene rps19' n'est pas exprime "in vivo" dans le chloroplaste en raison de la co-transcription sur l'autre brin des genes psba et trn h-gug. Les genes rp12 et rps19 codent respectivement pour les proteines ribosomiques chloroplastiques d'epinard l4 et s23 fortement homologues aux l2 et s19 d'e. Coli

Bien que l'initiation de la traduction soit un processus conservé entre les bactéries, nous avons montré que le mécanisme par lequel les ARNm structurés sont reconnus et adaptés sur le ribosome diffère chez Staphylococcus aureus, un micro-organisme avec un bas taux de G+C et chez Escherichia coli. Une particularité du ribosome de S. aureus est l'absence de la protéine ribosomale S1, qui non seulement est plus courte que celle de E. coli mais qui possède également une organisation distincte des domaines. Mes expériences suggèrent que la protéine S1 (SauS1) favorise spécifiquement l'initiation de la traduction de l'opéron α-psm 1-4 en liant son ARNm hautement structuré. En outre, il influence aussi l'expression et la production de facteurs de virulence comme les exotoxines (α-haemolysine, δ-hémolysine et γ- hémolysine) et les exoenzymes (protéases et lipases). En plus de son rôle dans la traduction, SauS1 pourrait être impliquée dans d'autres processus cellulaires tels que le métabolisme de l'ARN et la régulation par des ARN non-codants (ARNnc). Elle forme des complexes in vivo avec plusieurs ARNnc dont la stabilité serait affectée dans la souche délétée du gène rpsA codant S1. SauS1 a donc une activité chaperonne favorisant la cinétique d’appariement entre deux molécules d'ARN et au moins dans un cas, elle stimule la reconnaissance entre un ARNnc et son ARN cible. Ainsi, SauS1 appartient à une nouvelle classe de chaperons d'ARN qui jouent un rôle clé dans la régulation du virulon de S. aureus<br>Even if translation initiation is a conserved process among bacteria, we have recently shown that low G+C content Gram-positive, such as Staphylococcus aureus, differ from E. coli on the mechanism by which structured mRNAs are recognized and adapted on the ribosome. One peculiarity of the S. aureus ribosome is the absence of ribosomal protein S1, which is shorter than E. coli S1 and has different domains organization. My work could demonstrate that S. aureus S1 (SauS1) specifically promotes translation initiation of the α-psm 1-4 operon by binding its highly structured mRNA. Moreover, it influences the expression and production of other exotoxins (α-haemolysin, δ-haemolysin and γ-haemolysins) and exoenzymes (proteases and lipases). Besides its role in translation, SauS1 could be implicated in other cellular processes such as RNA maturation/degradation and sRNA-mediated regulation. It forms in vivo complexes with several sRNAs whose level is affected in a strain deleted of rpsA gene, coding for S1. Preliminary results show that SauS1 has a chaperone activity promoting the kinetic of annealing of two model RNA molecules and at least in one case, we could demonstrate that it stimulates the recognition between a sRNA and its target RNA. Taken together, SauS1 belongs to a new class of RNA chaperones that play key roles in the regulation of S.aureus virulon

Infectious bronchitis virus (IBV) is a member of family Coronaviridae and is classified into group 3 of the Coronaviruses. The virus is a single-stranded positive-sense RNA virus with a genome of 27kbp. IBV is a highly infectious disease of chickens that results in high morbidity with moderate to severe mortality depending on the strain involved, age of the birds, and immune status of the chickens. Multiple worldwide investigations indicate that differentiation within the S1 glycoprotein gene can lead to serotype variation within the IBV species. In this study 46 isolates collected over two years from broiler and broiler breeder flocks and eight historical isolates were analyzed. Forty one isolates originated from the KwaZulu-Natal region whilst the remaining thirteen were isolated from 4 other poultry-dense provinces. The S1 gene was sequenced and compared to determine variation between South African isolates, as well as global sequences submitted to Genbank. The results indicate the division of isolates analyzed into 2 different clades of IBV within the province. The most prevalent genotype was similar to IBV Mass strain detected in 79% of the full S1 sequences. Variation up to 22.3% was detected within local strains, supporting the hypothesis that multiple IBV serotypes may co-circulate in the same region simultaneously. Additionally, more conservation was observed among Mass serotypes versus QX-like serotypes, implying that vaccine use can influence the variability within the IBV population. Higher variability was found in the first half of the S1 gene in comparison to the last half of the S1 gene. This is in agreement with previous findings that the hypervariable regions of the S1 gene are located within the first 450 base pairs. This study offers the first published consolidation of IBV isolates from South Africa and identifies variation within the IBV population of the SA broiler flock. Previous publications list four or five IBV isolates whilst this study describes variation found in 54 isolates spanning 32 years. In addition this study provides the insight into the prevalence of IBV variation in poultry flocks due to the large number of isolates. The comparative use of geno- and serotyping for South African IBV isolates is also described for the first time in this study.<br>Dissertation (MSc)--University of Pretoria, 2013.<br>gm2014<br>Veterinary Tropical Diseases<br>unrestricted

La traduction est une étape clef de l’expression des gènes, et mon travail a consisté à étudier l’implication de la protéine ribosomique S1 d’Escherichia coli dans l’initiation de la traduction des ARNm structurés. Mes résultats montrent que 1) S1 est requise pour la formation du complexe d’initiation des ARNm portant une séquence SD faible et/ou des structures stables, 2) elle est dotée d’une activité chaperonne, débobinant les ARNm afin de les placer dans le canal de décodage ; et 3) le ribosome favorise son action. Par la suite, j’ai montré un rôle inattendu de S1 dans la régulation post-transcriptionnelle médiée par les ARNnc. En effet, la dégradation rapide de l’ARNm sodB, induite par l’ARNnc RyhB en absence de fer, est perdue dans une souche dont l’extrémité C-terminale de S1 a été supprimée, montrant ainsi un lien fonctionnel entre S1 et le dégradosome. Ainsi, S1 exerce de multiples fonctions qui se placent au carrefour de la traduction et de la régulation de l’expression des gènes<br>The translation is a key step for the gene expression, and the aim of my PhD was to analyze the involvment of Escherichia coli ribosomal protein S1 in the translation initiation of structured mRNAs.My results show that 1) S1 is required for the establishment of the active translation initiation complex involving mRNAs with a weak SD sequence and/or stable structures, 2) S1 has a RNA chaperone activity, unwinding the mRNA in order to accommodate it in the decoding channel, and 3) the ribosome promotes its activity.In the second part of my thesis, I unexpectedly showed that S1 is involved in the ncRNAmediated regulation. Indeed, the fast degradation of sodB mRNA, induced by RyhB ncRNA under iron depletion, is impaired in a strain depleted of the C-terminal part of S1 protein, thus highlighting a functional link between S1 and the degradosome.All in one, my results show that S1 is endowed with multiple functions, at the cross-road between translation and regulation of gene expression

A previously identified rat cDNA (S1) that shares 78% nucleotide homology and a predicted 92% amino acid sequence homology with human EF-1$ alpha$ was used to express S1 in E. coli and to generate a polyclonal antibody to pS1. A recombinant plasmid pGEX-2T-S1 was constructed, containing the glutathione S-transferase gene. The expressed fusion protein was purified and digested with thrombin to produce a recombinant S1 protein (rpS1) containing slightly modified N-terminus. Purified rpS1 was used to raise a rabbit antiserum which recognized rpS1 on immunoblots. A polyclonal antiserum to EF-1$ alpha$ failed to react with rpS1. Similarly the anti-rpS1 does not recognize EF-1$ alpha$ on immunoblots. Anti-rpS1 therefore is able to distinguish pS1 from EF-1$ alpha$ despite their extensive amino acid sequence homology. Anti-rpS1 and anti-Ef-1$ alpha$ will be used to study the similarities and differences between pS1 and EF-1$ alpha$ in vivo and in vitro.

Made available in DSpace on 2014-08-20T14:37:49Z (GMT). No. of bitstreams: 1 dissertacao_paula_finger.pdf: 729110 bytes, checksum: fe15bb22f0abb92bf1b2938fd6dcdfc9 (MD5) Previous issue date: 2011-12-19<br>The chickens infectious bronchitis (IB) is a highly contagious viral disease which causes predominantly respiratory lesions manifested clinically and invariably by sneezing and tracheo-bronchial rales, which may lead to more severe signs, with a decrease in fertility and reduction of eggs production. The infectious bronchitis virus (IBV) encodes four major structural proteins: N (nucleocapsid protein), S (spike protein), E (envelope protein) and M (membrane protein) being the S protein is cleaved into S1 and S2. The 1 subunit (S1) is found exposed in the viral envelope, which makes it an important or main inducer of neutralizing antibodies against the IBV, the main target for the host s immune system. The variations in two regions of the envelope s S1 subunit, called hypervariables regions, may originate to new serotypes. The IBV s mutation and recombination capacity and the selection pressure exerted by the prolonged use of live vaccines contribute to the appearance of a wide variety of serotypes and subtypes of IBV. The objective of this study was to express, in Pichia pastoris, the gene that encodes the surface protein S1 of IBV strain M41 and, in Escherichia coli, to express the S1 of the synthetic gene designed from consensus sequences of national and international field samples, as an interesting alternative for the production of antigen that can be used for monitoring vaccination of birds and also an antigen that is suitable for the use in serological diagnostic. The cloning and expression of glycoprotein S1 in both heterologous expression systems was successfully performed. The process of expression using E. coli was simple and quick when compared to the use of P. pastoris. The P. pastoris was able to express the entire S1; however, it showed difficulty in secreting the glycoprotein. The results will be evaluated for use in immunodiagnostic kit for monitoring the disease in poultry, being more affordable than the ones existing currently.<br>A bronquite infecciosa das galinhas (IB) é uma enfermidade viral altamente contagiosa que causa predominantemente lesões respiratórias que se manifestam clinicamente e invariavelmente por espirros e estertores tráqueo-bronquiolares, podendo levar a sinais mais severos, com diminuição na fertilidade e redução da produção de ovos. O vírus da bronquite infecciosa das galinhas (IBV) codifica quatro proteínas estruturais importantes: N (proteína do nucleocapsídeo), S (proteína de superfície), E (proteína do envelope) e M (proteína da membrana), sendo a proteína S subdividida em S1 e S2. A subunidade 1 (S1) encontra-se exposta no envelope viral, o que torna-a um importante, ou principal, indutor da produção de anticorpos neutralizantes frente ao IBV, sendo o principal alvo para o sistema imune do hospedeiro. As variações em duas regiões da subunidade S1 do envelope, chamadas regiões hipervariáveis, podem dar origem a novos sorotipos. A capacidade de mutação e recombinação de IBV e a pressão de seleção exercida pelo uso prolongado de vacinas vivas contribuem para o aparecimento de uma grande variedade de sorotipos e subtipos de IBV. O objetivo deste trabalho foi expressar em Pichia pastoris o gene que codifica a proteína de superfície S1 da estirpe M41 do IBV e, em Escherichia coli, expressar a S1 de um gene sintético elaborado a partir de sequências consenso de amostras de campo nacionais e internacionais, como uma alternativa interessante para a produção de antígeno que possa ser utilizado para monitoramento vacinal das aves e também um antígeno que seja adequado para utilização em diagnóstico sorológico. A clonagem e a expressão da glicoproteína S1 em ambos os sistemas heterólogos de expressão foi realizada com sucesso. O processo de expressão usando E. coli foi rápido e simples quando comparado ao uso da P. pastoris. A P. pastoris foi capaz de expressar a S1 inteira, porém, apresentou dificuldade em secretar a glicoproteína. Os resultados obtidos deverão ser avaliados para uso em Kit de imunodiagnóstico para monitoramento da enfermidade na avicultura, sendo de custo mais acessível do que os existentes no mercado.

Coronavírus, incluindo-se o Coronavírus aviário (ACoV), possuem o maior genoma composto por RNA conhecido entre os vírus. Aproximadamente dois terços desse genoma codificam proteínas não estruturais (Nsps), cujas funções parecem estar associadas à replicação e patogênese viral. Até o momento, esses alvos têm sido pouco explorados quanto a sua diversidade em diferentes linhagens de ACoV. O presente estudo teve como objetivo investigar a diversidade dos genes codificadores das proteínas não estruturais Nsp2 e protease Papaína-like (Plpro), utilizando-se linhagens brasileiras de ACoV. Para tanto, 10 linhagens de ACoV, isoladas em ovos embrionados, foram submetidas à RT-PCR direcionada aos genes codificadores de Plpro e Nsp2, seguindo-se o sequenciamento de DNA e a análise filogenética, juntamente com sequências homólogas obtidas no GenBank. Além disso, realizou-se a genotipagem por meio do sequenciamento parcial do gene codificador da proteína de espícula (região S1). Três das amostras virais obtidas e investigadas no presente trabalho apresentaram padrão de segregação discordante para os genes estudados. O isolado CRG I22 agrupou-se com linhagens virais pertencentes ao genótipo Massachusetts para S1 e com o grupamento de ACoVs brasileiros os genes da Nsp2 e Plpro. O isolado CRG I33 agrupou-se com linhagens virais pertencentes ao genótipo brasileiro para s1 e plpro e de maneira divergente para o gene da Nsp2. Para o isolado CRG I38, não foi obtida a genotipagem por s1, entretanto, similarmente ao observado para o isolado CRG I33, esse isolado agrupo-se com linhagens virais brasileiras para o gene plro e de maneira independente para o gene nsp2. As demais linhagens estudadas resultaram na formação de um grupamento especificamente brasileiro de ACoV, para os três genes estudados. Esses achados sugerem a ocorrência de recombinação nessas amostras discrepantes. Quanto às identidades médias entre as sequencias nucleotídicas analisadas, a região de s1 analisada apresentou as menores identidades (73,75% &plusmn;16,78), seguido pelo gene plpro (88,06% &plusmn;5,7) e do gene nsp2 (92,28% ±4,37), em acordo com a literatura. Assim sendo, os alvos investigados podem constituir ferramentas úteis na epidemiologia molecular do ACoV e na investigação de linhagens recombinantes do vírus. O presente estudo é o primeiro a investigar a diversidade genética de genes codificadores de proteínas não-estruturais em linhagens brasileiras de ACoV. Os resultados aqui apresentados reforçam a existência de um genótipo brasileiro de ACoV, para os 3 genes estudados. Entretanto, discrepâncias pontuais encontradas no padrão genotípico para s1, nsp2 e nsp3 permitem inferir uma diversidade genética maior do que a conhecida até o momento, possivelmente resultante de eventos de recombinação entre ACoVs brasileiros, ACoVs vacinais e outros ainda desconhecidos. Os resultados obtidos auxiliam na compreensão dos padrões e evolução dos ACoVs<br>Coronaviruses, including Avian coronavirus (ACoV), have the largest known RNA genome. Nearly two thirds of its genome codes for non-structural proteins (Nsps), whose functions appear to be linked to viral replication and pathogenesis. Hitherto these targets have been poorly explored regarding the ACoV lineages diversity. The present study aimed to assess the diversity of non-structural protein 2 (nsp2), papain-like protease (plpro) and spike protein (S1 subunit) coding genes, in Brazilian ACoV strains. To this end, 10 ACoV strains, isolated in embryonated eggs, had its 3rd and 5th passages submitted to RT-PCR targeting nsp2, plpro and s1, followed by DNA sequencing and phylogenetic analysis, herewith homologous sequences obtained from GenBank. Three of the ACoV strains sequenced showed a discordant segregation pattern for target genes. CRG I22 strain clustered with Massachusetts genotipe strains for S1, and with Brazilian cluster for nsp3 and plpro genes. CRG I33 strain, clustered with Brazilian strains for S1 and plpro genes, and was divergent for nsp2 gene. For CRG I38 strain, the S1 sequence was not obtained, however, similarly to what was observed for CRG I33, this strain grouped with the Brazilian lineage for plpro gene and was divergent for nsp2 gene. All the other ACoV here sequenced resulted in a specific Brazilian cluster for the three studied genes. Regarding the mean nucleotide identities measured, s1 gene showed the lowest identity (73.75% ±16.78), followed by plpro gene (88.06% ±5.7) and nsp2 gene (92.28% ±4.37), in accordance with previous reported data. Therefore, the targets of the present study are useful tools for ACoV molecular epidemiology studies and for the survey of recombinant ACoV strains. The presented study is the first one investigating the molecular diversity of non-structural proteins coding genes in Brazilian strains of ACoV. Results achieved herein reinforce the data over the circulation of ACoV Brazilian strains in this country, for the three investigated genes. However, divergences found between S1, nsp2 and plpro genetic patters allow inferring a higher molecular diversity than previously known. It is possible that this divergence is due to recombination events between ACoV from vaccines, Brazilian field strains and others still unknown. These results contribute on the comprehension over genetic patters and evolution of ACoV

Banana bunchy top virus (BBTV) causes one of the most devastating diseases of banana. Transgenic virus resistance is now considered one of the most promising strategies to control BBTV. Pathogen-derived resistance (PDR) strategies have been applied successfully to generate plants that are resistant to numerous different viruses, primarily against those viruses with RNA genomes. BBTV is a circular, single-stranded (css) DNA virus of the family Nanoviridae, which is closely related to the family Geminiviridae. Although there are some successful examples of PDR against geminiviruses, PDR against the nanoviruses has not been reported. Therefore, the aim of this thesis was to investigate the potential of BBTV genes to interfere with virus replication when used as transgenes for engineering banana plants resistance to BBTV. The replication initiation protein (Rep) of nanoviruses is the only viral protein essential for viral replication and represents an ideal target for PDR. Therefore, this thesis focused on the effect of wild-type or mutated Rep genes from BBTV satellite DNAs or the BBTV integral genome on the replication of BBTV in banana embryogenic cell suspensions. A new Rep-encoding satellite DNA, designated BBTV DNA-S4, was isolated from a Vietnamese BBTV isolate and characterised. When the effect of DNA-S4 on the replication of BBTV was examined, it was found that DNA-S4 enhanced the replication of BBTV. When the replicative capabilities of DNA-S4 and the previously characterised Rep-encoding BBTV satellite, DNA-S1, were compared, it was found that the amount of DNA-S4 accumulated to higher levels than DNA-S1. The interaction between BBTV and DNA-S1 was also examined. It was found that over-expression of the Rep encoded by DNA-S1 using ubi1 maize polyubiquitin promoter enhanced replication of BBTV. However, when the Rep-encoded by DNA-S1 was expressed by the native S1 promoter (in plasmid pBT1.1-S1), it suppressed the replication of BBTV. Based on this result, the use of DNA-S1 as a possible transgene to generate PDR against BBTV was investigated. The roles of the Rep-encoding and U5 genes of BBTV DNA-R, and the effects of over-expression of these two genes on BBTV replication were also investigated. Three mutants of BBTV DNA-R were constructed; plasmid pUbi-RepOnly-nos contained the ubi1 promoter driving Rep expression from DNA-R, plasmid pUbi-IntOnly-nos contained the ubi1 promoter driving expression of the DNA-R internal gene product (U5), while plasmid pUbi-R.ORF-nos contained the ubi1 promoter driving the expression of both Rep and the internal U5 gene product. The replication of BBTV was found to be significantly suppressed by pUbi-RepOnly-nos, weakly suppressed by pUbi-IntOnly-nos, but strongly enhanced by pUbi-R.ORF-nos. The effect of mutations in three conserved residues within the BBTV Rep on BBTV replication was also assessed. These mutations were all made in the regions in the ATPase motifs and resulted in changes from hydrophilic to hydrophobic residues (i.e. K187→M, D224→I and N268→L). None of these Rep mutants was able to initiate BBTV replication. However, over-expression of Reps containing the K187→M or N268→L mutations significantly suppressed the replication of BBTV. In summary, the Rep constructs that significantly suppressed replication of DNA-R and -C in banana embryogenic cell suspensions have the potential to confer resistance against BBTV by interfering with virus replication. It may be concluded that BBTV satellite DNAs are not ideal for conferring PDR because they did not suppress BBTV replication consistently. Wild-type Rep transcripts and mutated (i.e. K187→M and N248→L) Rep proteins of BBTV DNA-R, however, when over-expressed by a strong promoter, are all promising candidates for generating BBTV-resistant banana plants.

碩士<br>國立中興大學<br>微生物暨公共衛生學研究所<br>100<br>The cytokines are naturally derived proteins that play important roles in controlling and promoting immune responses, and therefore may have potential to serve as good vaccine adjuvants. Avian infectious bronchitis (IB) is an important avian disease and its causative agent is avian infectious bronchitis virus (IBV). Many different IBV serotypes have been recognized with little cross protection, rendering great economic losses due to vaccination failure. In this study, we expressed fusion cytokines and S1 subunit of IBV for the future vaccine usage. To produce fusion cytokines, we linked A protein with C or B protein by a 10 amino acid (flexible peptide linker (S2 linker). Using pET32a vector, we constructed expression plasmids for A-S2-C and A-S2-B. After protein induction and purification, both S2 linker-derived recombinant proteins were compared in parallel with αH linker (15-amino acid helix forming linker) derived proteins, which were developed earlier in our laboratory. Activity assays were conducted to evaluate the activities of recombinant cytokines. The results showed that αH linker derived cytokines were better than S2 linker-derived cytokines in stimulating lymphocyte proliferation or inducing nitric oxide production from macrophages. S2 linked cytokine showed a stable lymphocyte proliferation stimulating activity in a wide range of protein concentration. For the expression of IBV S1 subunit proteins, we amplified the entire or partial S1 gene fragments from a vaccine strain (H120) and a field strain (TW1), and constructed 6 prokaryotic expression plasmids. After induction, bacteria carrying partial S1 gene fragments expressed recombinant proteins with the expected molecular weights. These IBV S1-derived proteins will be evaluated together with fusion cytokines as immunogen and adjuvant for the vaccine usage.

abstract: Transient receptor potential vanilloid member 1 (TRPV1) is a membrane protein ion channel that functions as a heat and capsaicin receptor. In addition to activation by hot temperature and vanilloid compounds such as capsaicin, TRPV1 is modulated by various stimuli including acidic pH, endogenous lipids, diverse biological and synthetic chemical ligands, and modulatory proteins. Due to its sensitivity to noxious stimuli such as high temperature and pungent chemicals, there has been significant evidence that TRPV1 participates in a variety of human physiological and pathophysiological pathways, raising the potential of TRPV1 as an attractive therapeutic target. However, the polymodal nature of TRPV1 function has complicated clinical application because the TRPV1 activation mechanisms from different modes have generally been enigmatic. Consequently, tremendous efforts have put into dissecting the mechanisms of different activation modes, but numerous questions remain to be answered. The studies conducted in this dissertation probed the role of the S1-S4 membrane domain in temperature and ligand activation of human TRPV1. Temperature-dependent solution nuclear magnetic resonance (NMR) spectroscopy for thermodynamic and mechanistic studies of the S1-S4 domain. From these results, a potential temperature sensing mechanism of TRPV1, initiated from the S1-S4 domain, was proposed. Additionally, direct binding of various ligands to the S1-S4 domain were used to ascertain the interaction site and the affinities (Kd) of various ligands to this domain. These results are the first to study the isolated S1-S4 domain of human TRPV1 and many results indicate that the S1-S4 domain is crucial for both temperature-sensing and is the general receptor binding site central to chemical activation.<br>Dissertation/Thesis<br>Doctoral Dissertation Biochemistry 2019

碩士<br>國立臺灣大學<br>微生物學研究所<br>86<br>Hepatitis B viral infection, like other viruses, it should first bind to the r eceptor(s) on the target cell surface by its envelope protein and the membrane fusion will occur between HBV and target cell. After entering the cell, it wi ll begin its replication. So successful infection of HBV depends on whether HB V could bind the receptor(s) on the target cell. It is not yet clear what is H BV receptor. There are data showing that the HBV large surface antigen (L) is involved in the viral-cell interaction. The pre-S1 region of L binds to the re ceptor(s) and the amino acid residues to 47 in the pre-S1 region may be the s pecific binding site. The pre-S2 may help and increase the binding efficiency. We tried to find the HBV receptor(s) by the RAP in situ method. Two kinds of p re-S1-alkaline phosphatase (AP) fusion proteins, N-terminal insertion and C-te rminal insertion were constructed. These two fusion proteins had different mol ecular weight and different biological functions. As experiments found, the di fference in molecule weight was partially due to the glycosylation pattern bet ween these fusion proteins. Both the constructs could bind to the hepatocytes on the frozen liver tissue section but with different efficiency. The binding could be partially blocked by the pre-S1, suggesting that this binding is spec ific. Further modification will make the fusion proteins as useful probes in t he searching for HBV receptor(s).By fusing the putative receptor binding domai n (pre-S1) of HBV envelope protein to the reporter molecular (in this case is human placenta alkaline phosphatase), we could use the chimera proteins to ide ntify its cellular binding counterparts (may be receptors).

碩士<br>國立中興大學<br>分子生物學研究所<br>103<br>Canine coronavirus (CCoV), Canine parvovirus (CPV) and Leptospira interrogans, All are common pathogens for canine. CCoV　and CPV can cause gastrointestinal disease of dogs. CPV will lead to canine respiratory disease and the Leptospira can affect liver and kidney function and cause internal bleeding and gastrointestinal diseases according to their different serotypes. CCoV enters the cell utilize spike protein. CPV VP2 protein is important role into host cells. Leptospirosis is use Lig.A and Lig.B proteins into host cells. In this study, we product protein by BL21 (DE3), After the fusion protein was purified immunized BALB / c mice, Whichever spleen cells with myeloma cells (NS1) for cell fusion, use enzyme-linked immunosorbent assay (ELISA) Preliminary screening antibody secreting tumor cell fusion, And TrxA-His and GST negative control group was further confirmation may identify specific antigen positive. After confirming via we were selected from two CCoV S1 protein of the monoclonal antibody, we named S1-35 and S1-38. In Lig.B Leptospira proteins have also been two monoclonal antibodies, named Lig.B-1 and Lig.B-2. Use of western blot and immune blot analysis showed that the S1-35 and S1-38 which identify antigens are non-configuration-dependent (linear antigen)；Lig.B-1 and Lig.B-2 which recognize the same antigen non-conformation-dependent decision bits. In Pierce ™ Rapid Antibody Isotyping Kit-Mouse (Thermo) antibody subtype analysis kit, S1-35 and S1-38 two monoclonal antibodies are all of its antibodies in IgG1, the same as the antibody light chain κ, The Lig.B-1 and Lig.B-2 two monoclonal antibodies for antibody subtype IgG1, the same as the antibody light chain κ. Similarly, to remove the mutation (deletion mutation) positioning mode antigenic determinant bit of analysis, Monoclonal antibodies S1-35 recognize S1 170-262 amino acids and monoclonal antibodies S1-38 recognize S1 262-353 amino acids, Lig.B-1 monoclonal antibody identified LigB 301-598 amino acids and monoclonal antibodies identify Lig.B 1-300 amino acids. Over the past years our laboratory Nian Lun Zheng locate CPV-VP2 epitopes bit of analysis prepared, The results showed that two strains of CPV-VP2 monoclonal antibody to identify the location of epitopes bit of VP2 protein were first and second amino acids 85-97 amino acids 52-62. Preparation of monoclonal antibodies identified in this study can be used in addition to antigen subunit vaccine experiments dogs, the future may further be used as in Immunochromatographic assay (ICT) of the material, for rapid pathogen detection.