ABSTRACT
Homogeneous (according to PAGE) capsid and surface viral proteins were isolated from concentrated purified suspensions of tick-borne encephalitis and Venezuelan equine encephalomyelitis viruses by one-stage reversed-phase HPLC. The amino acid composition and the sequences of their N-terminal parts were determined.
Subject(s)
Encephalitis Virus, Venezuelan Equine/chemistry , Encephalitis Viruses, Tick-Borne/chemistry , Viral Structural Proteins/isolation & purification , Chromatography, High Pressure Liquid , Viral Structural Proteins/chemistryABSTRACT
Foreign DNA fragments were inserted into filamentous phage gene VIII to create hybrid B-proteins with foreign sequences in the amino terminus. The hybrid proteins are incorporated into the virions which retain viability and infectivity. Virions with hybrid B-proteins have the same contour length and the same number of B-protein molecules as virions with natural B-proteins. It was shown that for one of hybrid B-proteins the position of the processing site had changed.
Subject(s)
Bacteriophages/genetics , Capsid/genetics , Mutagenesis, Site-Directed , Peptides/genetics , Amino Acid Sequence , Bacteriophages/pathogenicity , Base Sequence , Molecular Sequence Data , PlasmidsSubject(s)
Escherichia coli/metabolism , Genes, Synthetic , Interferon-alpha/genetics , Peptide Biosynthesis , Amino Acid Sequence , Amino Acids/analysis , Base Sequence , Chromatography, High Pressure Liquid , Gene Expression , Humans , Hydrolysis , Molecular Sequence Data , Peptides/metabolism , Plasmids , Sequence Homology, Nucleic AcidABSTRACT
Constituent proteins of human influenza virus A have been separated by reverse-phase HPLC on Polysil ODS-500. Their homogeneity is confirmed by the data of amino acid composition, Edman analysis and gel electrophoresis.
Subject(s)
Influenza A virus/metabolism , Viral Proteins/isolation & purification , Amino Acid Sequence , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Serum Albumin, Bovine/isolation & purification , Viral Proteins/geneticsABSTRACT
M13B1 vector based on the filamentous phage M13 has been constructed. M13B1 phage carries the gene of resistance to ampicillin and contains the unique site of recognition for BamHI restriction endonuclease in gene VIII coding for the major coat protein. BamHI restriction site has been inserted into the gene of the major coat protein by means of oligonucleotide directed mutagenesis. The synthetic DNA fragment coding for the model peptides has been inserted through BamHI site into the M13B1 DNA. The possibility of inserting foreign peptides into the N-terminus at maintaining the viability of hybrid phages has been shown. The differences in specificity of the recombinant phage maturation have been determined by analysing the amino acid sequence of B-protein.
Subject(s)
Capsid Proteins , Capsid/genetics , Coliphages/genetics , DNA, Viral/genetics , Membrane Proteins/genetics , Protein Engineering , Amino Acid Sequence , Base Sequence , Blotting, Southern , Molecular Sequence Data , Mutation , Recombination, GeneticABSTRACT
The hybrid gene of influenza virus hemagglutinin (HA) of the H1-subtype, carrying the sequence coding for the fragment of H3-subtype antigenic site B, was constructed. The product of expression of this gene in E. coli was obtained as a fusion protein with beta-galactosidase. The chimeric protein was shown to retain the antigenic properties of HA of H1-subtype and to interact specifically with antibodies against the synthetic peptide corresponding to the B site fragment of HA of the H3-subtype.
Subject(s)
Gene Expression , Genes, Synthetic , Hemagglutinins, Viral/biosynthesis , Influenza A virus/immunology , Recombinant Proteins/biosynthesis , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Epitopes/immunology , Escherichia coli/genetics , Genes, Viral , Hemagglutinins, Viral/genetics , Hemagglutinins, Viral/immunology , Influenza A virus/genetics , Molecular Sequence Data , Protein Conformation , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
The full-length copy of the hemagglutinin gene of influenza virus was inserted into M13 phage DNA. The DNA sequence coding for the hydrophobic prepeptide was removed from the gene by oligonucleotide-directed mutagenesis. The possibilities of expression of the full-length and mutant genes in E. coli were investigated. The beta-galactosidase-hemagglutinin fusion proteins were isolated. The fusion proteins exhibited specific binding to antiviral antibodies. This binding could be competitively inhibited by excess of viral hemagglutinin, demonstrating that these fusion proteins contained antigenic determinants of hemagglutinin.
Subject(s)
Escherichia coli/genetics , Gene Expression Regulation , Hemagglutinins, Viral/genetics , Influenza A virus/genetics , Bacteriophages/genetics , Cloning, Molecular , DNA/drug effects , Electrophoresis, Polyacrylamide Gel , Genes, Viral , Plasmids , Radioimmunoassay , Restriction Mapping , Viral Fusion Proteins/genetics , Viral Fusion Proteins/isolation & purificationABSTRACT
The receptor properties of influenza virus A/Kiev/59/79 R (H1N1) and a number of its polypeptide fragments containing the aminoacids (from the 1st to 272d) of the heavy chain were studied. Two kinds of radioimmunoassay were used to test hemagglutinin or its polypeptide fragment interactions with cellular receptors. The studied polypeptides and hemagglutinin are shown to be capable of specific interactions with the receptors on the cell surface. The main linear fragment of hemagglutinin recognizing cellular receptors is localized within a polypeptide fragment including 1st-272d aminoacids of the heavy chain of hemagglutinin. The breaks of all the the S-S linkages including the ones linearly and spatially close to the receptor "pocket" of the bridge 95-135 do not affect significantly the receptor properties of the polypeptide.
