RESUMO
Infectious bursal disease virus(IBD) causes infectious bursal disease (IBD), which infects bursal of chicken and can evoke immune suppression. This study identified the antigenic epitopes of four McAbs to IBDV VP3(HRB-3F, HRB-7B, HRB-7C and HRB-10E)with pepscan. A set of 17 partially overlapping or consecutive peptides (P1-P17) spanning VP3 were expressed for epitope screening by pepscan. Finally, two antigenic epitopes, 109-119aa and 177-190aa of IBDV VP3, were identified by Western blot and ELISA. The peptides on epitopes could react with IBDV, and they had better immunnogenicity. The sequences of epitopes were compared with that of several other IBDV strains in the same region, and was found they were totally homologous. This study showed the two epitopes were novel conserved linear B cell epitopes on the VP3 of IBDV. This study provides basis for the development of immunity-based prophylactic, therapeutic and diagnostic measures for control of IBD and further for structural and functional analysis of IBDV.
Assuntos
Animais , Camundongos , Anticorpos Monoclonais , Alergia e Imunologia , Anticorpos Antivirais , Sangue , Alergia e Imunologia , Western Blotting , Proteínas do Capsídeo , Genética , Alergia e Imunologia , Metabolismo , Ensaio de Imunoadsorção Enzimática , Epitopos , Genética , Alergia e Imunologia , Metabolismo , Soros Imunes , Alergia e Imunologia , Imunização , Imuno-Histoquímica , Vírus da Doença Infecciosa da Bursa , Genética , Alergia e Imunologia , Metabolismo , Camundongos Endogâmicos BALB CRESUMO
OBJECTIVE@#To identify the promoter of human nicastrin (NCT) gene, a major component of gamma-secretase which is closely related with pathogenesis of Alzheimer's disease.@*METHODS@#Promoter of human Alzheimer's disease related gene, nicastrin, a 1768 bp fragment was firstly isolated from human genomic DNA by PCR. This fragment's 3 flanking end was 4 bp upstream to the start codon ATG (+1) of the gene. This fragment was used as template, a series of deleted fragments were amplified and constructed to the pGL3-Enhancer plasmid with the artificial designed linkers. The relative activity of their promoter in Hela cells was studied by dual-luciferase assay.@*RESULTS@#The 420 bp fragment showed the strongest activity, and the 237 bp fragment was the minimal fragment in length with activity.@*CONCLUSION@#The promoter of NCT is located at -432/-133 region upstream the translational start codon, while its basal promoter is between -359/-90 that drives the transcription of reporter gene in Hela cells.
Assuntos
Humanos , Doença de Alzheimer , Genética , Secretases da Proteína Precursora do Amiloide , Clonagem Molecular , Genes Reporter , Genética , Células HeLa , Glicoproteínas de Membrana , Genética , Regiões Promotoras Genéticas , GenéticaRESUMO
<p><b>OBJECTIVE</b>To construct a phosphatidylinositol 4-kinase beta (PI4K-beta) mutant with the 325th to 373rd amino acid codons deleted, and try to develop a simple method for constructing middle fragment deletion mutant.</p><p><b>METHODS</b>In line with the mechanism of gene splicing by overlap extension(SOE), an additional PCR was used to get the PI4K-beta mutant in which the 325th to 373rd amino acid codons were deleted. Then the mutated gene was cloned into pCMV-Tag4A mammalian expression vector.</p><p><b>RESULTS</b>A mutant with the 325th to 373rd amino acid codons deleted was constructed successfully.</p><p><b>CONCLUSION</b>The improved SOE is a very effective and reliable method to construct middle fragment deletion mutant. It is worthy to be popularized.</p>