Non-structural protein, NS1 of African horse sickness virus is a hydrophobic protein of 63 kDa that spontaneously assembles into highly distinct tubular structures when expressed in mammalian or insect cells. The spontaneous assembly of these proteins into a predictable multimeric structure, high levels of expression and ease of purification make this protein an ideal candidate for the immune display of foreign peptides. The potential of such a display system has been investigated for BTV NS1 that is able to successfully elicit both a humoral and a cellular immune response against inserted peptides. The aims of this study were to investigate both the stability of the AHSV NS1 particulate structure after insertion of peptides as well as the antigenicity and immunogenicity of the peptides presented in this system. Two overlapping regions consisting of 40 and 150 amino acids, and which correspond to a neutralising region identified within the AHSV major neutralising protein VP2, were inserted into an internal site in NS1. This site offered the best surface display of inserted peptides on the tubular structures. An enhanced green fluorescent protein, 240 amino acids long, was also inserted into the NS1 protein. Sucrose gradient analysis of the recombinant proteins indicated that the majority of the baculovirus expressed chimeric proteins formed particulate structures with a sedimentation value similar to that of the native NS1 protein. This was confirmed by transmission electron microscopic analysis, which clearly showed that all the chimeric proteins assembled into tubular structures similar to those observed for AHSV NS1 proteins. Furthermore, fluorescence analysis of sucrose gradients of NS1/eGFP also showed high levels of fluorescence that corresponded directly to particle formation. Not only do the inserts remain functional but are also presented successfully on the surface of the intact NS1 tubule structure. The potential of the NS1 vector to efficiently present peptides to the immune system was subsequently investigated. The serums generated against these chimeric proteins in guinea pigs were tested against chimeric constructs, the baculovirus expressed inserts (for eGFP) and the inserts presented on other presentation vectors. Western blot analysis showed that most of the serums generated against the chimeric proteins contained antibodies not only against the chimeric proteins but antibodies that reacted specifically with the inserted peptides on their own or on another presentation system. Preliminary immune studies seem to indicate that the humoral immune response elicited by the chimeric NS1 proteins is predominantly against the inserts. The inserts are successfully presented to the immune system on the surface of the NS1 vector and are able to elicit the production of antibodies with the potential to provide a protective immune response. / Dissertation (MSc (Genetics))--University of Pretoria, 2010. / Genetics / unrestricted

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-03-06T13:55:03Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_mariana_peil_da_rosa.pdf: 780579 bytes, checksum: 32fb27eef72fa6d21ed64bfe1f7c5af1 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-03-09T20:15:32Z (GMT) No. of bitstreams: 2 dissertacao_mariana_peil_da_rosa.pdf: 780579 bytes, checksum: 32fb27eef72fa6d21ed64bfe1f7c5af1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-03-09T20:17:03Z (GMT) No. of bitstreams: 2 dissertacao_mariana_peil_da_rosa.pdf: 780579 bytes, checksum: 32fb27eef72fa6d21ed64bfe1f7c5af1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-09T20:17:13Z (GMT). No. of bitstreams: 2 dissertacao_mariana_peil_da_rosa.pdf: 780579 bytes, checksum: 32fb27eef72fa6d21ed64bfe1f7c5af1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-03-31 / Sem bolsa / O objetivo deste trabalho foi avaliar a expressão do gene HPS no tegumento de quatro genótipos de soja, em épocas distintas de coleta após a antese. O experimento foi conduzido em casa de vegetação da Estação Terras Baixas da Embrapa Clima Temperado e no Laboratório de Sementes e Biotecnologia (UFPel/FAEM), em delineamento completamente casualizado, com três repetições. Utilizou-se duas cultivares de tegumento amarelo, COODETEC 202 e BMX Potência RR (POT) e duas linhagens de tegumento preto, Tegumento Preto (TP) e IAC 222. Efetuou-se sete coletas dos tegumentos, em intervalos de 5 dias cada, durante o desenvolvimento das sementes de soja entre os períodos de 25 a 55 dias após a antese. Procedeu-se a extração de RNA dos tegumentos, seguido da obtenção do cDNA para posterior análise de qRT-PCR, visando quantificar o acúmulo relativo do gene HPS. A cultivar IAC 222 apresentou valores de quantificação relativa superior em todas as épocas avaliadas, sendo o maior valor encontrado na coleta de 35 dias após a antese. O acúmulo relativo de transcritos do gene HPS no tegumento de sementes de soja difere entre as épocas avaliadas, bem como entre os genótipos estudados. / The objective of this study was to evaluate the gene expression of HPS in the seed coat of four soybean genotypes at different sampling times after anthesis. The experiment was conducted in greenhouse at Estação Terras Baixas of Embrapa Clima Temperado and Laboratório de Sementes e Biotecnologia (UFPel / FAEM) in Capão do Leão city - RS, in a randomized complete block design with three replicates, and treatments arranged in a factorial model. Were used four contrasting genotypes, two cultivars of yellow coat (COODETEC 202 and BMX Potência RR) and two genotypes of black coat (Tegumento Preto and IAC). Seed coat samples were collected along the developing soybean seed, intervals for 5 days, between the periods 25-55 days after anthesis. Proceeded the RNA extraction from the seed coats, followed by obtaining the cDNA for further analysis of qRT-PCR, to quantify the relative accumulation of gene transcripts HPS. IAC 222 showed higher values of relative quantification in all periods, with the highest value found in the collection of 35 days after anthesis. The relative accumulation of transcripts of the HPS gene in the seed coat of soybean differs between the periods evaluated, as well as between genotypes.