RESUMO
For a long time, many types of vaccines have been useful for the prophylaxis of many infectious diseases. Thus far, many adjuvants that enhance the effects of vaccines have been explored. However, very few adjuvants are being used for humans worldwide. In this study, we investigated the adjuvant activity of various substances, and found citrulline to have high potential as an adjuvant. Citrulline is a type of amino acid present in the body of many organisms. A number of biological activities of citrulline have been reported; however, no adjuvant activity has been reported thus far. Aluminum salts, which are commonly used as adjuvants are not water soluble; therefore, some difficulties are encountered while using them as vaccine adjuvants. Citrulline is easy to use because of its water solubility. In this study, we showed for the first time the adjuvant activity of citrulline by using viral antigens and amyloid ß peptide. Water-soluble citrulline, which is present in our body, is a potential adjuvant candidate.
Assuntos
Adjuvantes Imunológicos , Citrulina , Vacinas , Compostos de Alúmen , Peptídeos beta-Amiloides/imunologia , Animais , Arginina/imunologia , Citrulina/imunologia , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunização , Camundongos , Peptídeos/imunologia , Vacinas/imunologia , Vacinas de Produtos InativadosRESUMO
For the development of a safe vaccine for Alzheimer's disease (AD), we studied the immunogenicity of amyloid beta (Abeta) peptides without adjuvant. Addition of a cysteine residue (Cys) to Abeta peptides enhanced immunogenicity in mice compared to those without Cys. Vaccination with the Abeta-Cys peptides reduced Abeta deposits in AD model mice. From these results, the Abeta-Cys peptides, administered without adjuvant, are considered candidates for vaccine therapy for AD.
Assuntos
Doença de Alzheimer/terapia , Vacinas contra Alzheimer/imunologia , Peptídeos beta-Amiloides/imunologia , Cisteína/imunologia , Adjuvantes Imunológicos/administração & dosagem , Doença de Alzheimer/imunologia , Vacinas contra Alzheimer/administração & dosagem , Sequência de Aminoácidos , Peptídeos beta-Amiloides/administração & dosagem , Animais , Encéfalo/imunologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , VacinaçãoRESUMO
We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.
Assuntos
Endopeptidases/química , Precursores Enzimáticos/metabolismo , Protrombina/metabolismo , Proteínas Recombinantes/química , Actinas/metabolismo , Animais , Biotecnologia/métodos , Western Blotting , Células CHO , Bovinos , Linhagem Celular , Galinhas , Cromatografia de Afinidade , Cricetinae , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Precursores Enzimáticos/isolamento & purificação , Vetores Genéticos , Humanos , Cinética , Metotrexato/farmacologia , Camundongos , Mieloma Múltiplo/metabolismo , Mutação , Plasmídeos/metabolismo , Agregação Plaquetária , Regiões Promotoras Genéticas , Protrombina/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Sefarose/química , Tetra-Hidrofolato Desidrogenase/genética , Trombina/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Megakaryoblastoma (Dami cells) cultured in a serum-free medium containing albumin, proliferated for three days but died on the fourth day. This cell death was not observed when human plasma was added, suggesting that human plasma contains a cell-death inhibitory factor. In order to identify this factor, we purified it from human plasma. N-terminal amino acid sequence analysis revealed that this factor is a mixture of C-terminal fragments of selenoprotein P, a major selenocysteine-containing protein in plasma. The specific activity (unit per pmol of selenium) of selenoprotein P fragments protein was 15-fold and 1900-fold higher than that of the full-length SeP and sodium selenite, respectively.