RESUMO
We have investigated the use of soluble chimeric trimers of the major capsid protein VP7 of African horse sickness virus (AHSV) as a vaccine delivery system by targeting some of the natural hydrophilic loops on the VP7 top domain for the insertion of foreign peptides. Key to this trimer display strategy is the solubility of AHSV VP7 and how the solubility of this hydrophobic protein can be manipulated by inserting peptides into the top domain. To investigate, we generated different cloning vectors by inserting multiple cloning sites at three different positions in the VP7 gene. These modifications inserted six amino acids at the cloning sites and in some cases this converted VP7 to a largely soluble protein without affecting the ability of the modified proteins to form trimers. The vectors were used to generate a number of soluble VP7 fusion proteins including a fusion with a 36 amino acid insert that overlaps important immunological domains on protein VP1 of foot and mouth disease virus (FMDV) as well as a 110 amino acid peptide derived from AHSV VP2. Soluble trimers of these fusion proteins were able to elicit a good insert-specific immune response in guinea pigs. l-Arginine was found to reverse protein aggregation and was employed as an effective strategy to isolate relatively pure soluble chimeric VP7 trimers. Another factor that increased VP7 solubility in both wild-type VP7 and one of the VP7 vector proteins was the substitution of the leucine residue in position 345 of the VP7 C-terminus with a hydrophilic arginine residue.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos Virais/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas do Core Viral/imunologia , Substituição de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Antígenos Virais/química , Antígenos Virais/genética , Arginina/metabolismo , Baculoviridae/genética , Feminino , Vetores Genéticos/genética , Cobaias , Imunidade Humoral/imunologia , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Solubilidade , Proteínas do Core Viral/química , Proteínas do Core Viral/genéticaRESUMO
Numerous gene-specific PCR methods have been developed for the cultivation-independent discovery of novel genes from complex environmental DNA samples. The recovery of full-length genes is, however, technically challenging. Here, we present an efficient and relatively simple approach that combines magnetic bead capture with subtractive hybridization for the rapid and direct recovery of full-length target ORFs. When compared with other PCR-based techniques, a higher degree of specificity is achieved through the use of larger gene fragments during hybridization followed by several high-stringency washes. Together with the recent advances in environmental nucleic acid extraction techniques, this approach should allow for the further exploration of the metagenomic sequence space.
Assuntos
DNA/isolamento & purificação , Separação Imunomagnética , Hibridização de Ácido Nucleico , Fases de Leitura Aberta , DNA/análise , Separação Imunomagnética/instrumentação , Separação Imunomagnética/métodos , Metagenoma , Metagenômica/instrumentação , Metagenômica/métodos , Hibridização de Ácido Nucleico/métodos , Fases de Leitura Aberta/genética , Sensibilidade e EspecificidadeRESUMO
A new method for the recovery of full-length open reading frames from metagenomic nucleic acid samples is reported. This technique, based on subtractive hybridization magnetic bead capture technology, has the potential to access multiple gene variants from a single amplification reaction. It is now widely accepted that classical microbiological methods provide only limited access to the true microbial biodiversity (less than 1%). The desire to access a higher proportion of the metagenome has led to the development of efficient environmental nucleic acid extraction technologies and to a range of sequence-dependent and sequence-independent gene discovery techniques. These methods avoid many of the limitations of culture-dependent gene targeting.
Assuntos
Mapeamento Cromossômico/métodos , DNA/genética , DNA/isolamento & purificação , Hibridização In Situ/métodos , Magnetismo , Fases de Leitura Aberta/genética , Análise de Sequência de DNA/métodos , Marcação de Genes/métodos , Genômica/métodos , MicroesferasRESUMO
It is now widely accepted that the application of standard microbiological methods for the recovery of microorganisms from the environment has had limited success in providing access to the true extent of microbial biodiversity. It follows that much of the extant microbial genetic diversity (collectively termed the metagenome) remains unexploited, an issue of considerable relevance to a wider understanding of microbial communities and of considerable importance to the biotechnology industry. The recent development of technologies designed to access this wealth of genetic information through environmental nucleic acid extraction has provided a means of avoiding the limitations of culture-dependent genetic exploitation.
