ABSTRACT
The results of the study of the structure and function of harpin-like peptides (alpha-harpinins) of the EcAMP group from the barnyard grass (E. crusgalli) seeds and the possibility of their involvement in the innate immunity to biotic stresses are presented.
Subject(s)
Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Echinochloa/chemistry , Phytophthora infestans/growth & development , Plant Proteins/chemistry , Plant Proteins/pharmacology , Protein Structure, Secondary , Structure-Activity RelationshipABSTRACT
This is the first report describing the possibility of using a green fluorescent protein chromophore synthetic analog, P-HOBDI-BF2, as a fluorescent dye for a linear hydrolysis probe used in qPCR. The study was carried out on a system for detection of the plant pathogenic fungus Fusarium avenaceum using a plasmid containing translation elongation factor 1α fragment as a template. To estimate fluorogenic properties of P-HOBDI-BF2, 6-FAM- and BDP-FL-labeled probes were used. It was demonstrated that a synthetic dye based on the P-HOBDI-BF2 chromophore can be used for labeling hydrolysis probes for qPCR, but fluorescence increase levels for P-HOBDI-BF2-labeled probes were slightly lower than those for 6-FAM-labeled ones. At the same time, the sensitivity of P-HOBDI-BF2-based assays remained high, and this fact together with acceptable fluorescence levels suggests that this dye can be considered as an efficient alternative for reporters traditionally used for fluorescence detection in the FAM channel.
Subject(s)
Fluorescent Dyes/chemistry , Green Fluorescent Proteins/chemistry , Real-Time Polymerase Chain Reaction/methods , Base Sequence , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fusarium/genetics , Imidazoles/chemistryABSTRACT
Successful disease prevention and therapy critically depend on timely diagnosis of infections. Quantitative immuno-PCR (qiPCR) technology improves the sensitivity in the detection of antibodies to pathogens. A qiPCR-based assay was developed to determine IgG antibodies to Epstein-Barr virus (EBV) in the human blood serum. EBV nuclear protein 1 fragment (pEBV) was expressed in Escherichia coli. A synthetic single-stranded deoxyribonucleotide was conjugated to streptavidin, and the conjugate was used to detect ÑEBV-IgG1-biotin complexes by qiPCR. The IgG1 titers determined by qiPCR were compared to the results of enzyme-linked immunosorbent assay (ELISA). The sensitivity of qiPCR was one order of magnitude higher than that of ELISA. Thus, a highly sensitive qiPCR-based assay was developed to quantitate antibodies specific to the recombinant EBV antigen.
Subject(s)
Antibodies/blood , Enzyme-Linked Immunosorbent Assay/methods , Epstein-Barr Virus Infections/blood , Herpesvirus 4, Human/isolation & purification , Antigens, Viral/blood , Antigens, Viral/immunology , Epstein-Barr Virus Infections/immunology , Herpesvirus 4, Human/immunology , Herpesvirus 4, Human/pathogenicity , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Nuclear Proteins/blood , Nuclear Proteins/immunology , Polymerase Chain ReactionABSTRACT
Genes encoding two three-finger toxins TFT-AF and TFT-VN, nucleotide sequences of which were earlier determined by cloning cDNA from venom glands of vipers Azemiops feae and Vipera nikolskii, respectively, were expressed for the first time in E. coli cells. The biological activity of these toxins was studied by electrophysiological techniques, calcium imaging, and radioligand analysis. It was shown for the first time that viper three-finger toxins are antagonists of nicotinic acetylcholine receptors of neuronal and muscle type.
