ABSTRACT
The most potent antigen among HSV-1 proteins are glycoproteins gB(UL27) and gD(US6). Multiple amino acid sequence alignment of these proteins shows that gD protein is the most specific for HSV-1. Analysis of gD protein epitopes detected the main antigenic determinants not cross-reactive with antigens of other viruses. Virus was isolated and genome DNA was prepared from morphological elements of a patient with herpes simplex infection. US6 gene fragment was cloned in pUC19 vector. Cloning in bacterial expression vectors helped obtain beta-galactosidase-fused recombinant HSV-1 gD protein with 6-histidines affine target for high-performance chromatography purification. ELISA with a set of HSV-1-positive and negative donor sera and a commercial panel of HSV-1 sera (Vektor-Best) showed that recombinant gD can be used as an antigen to HSV-1-specific IgG.
Subject(s)
Epitopes/chemistry , Viral Envelope Proteins/chemistry , Amino Acid Sequence , Chromatography, High Pressure Liquid , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Epitopes/genetics , Epitopes/isolation & purification , Genetic Vectors , Humans , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sequence Homology, Amino Acid , Viral Envelope Proteins/genetics , Viral Envelope Proteins/isolation & purificationABSTRACT
Analysis of published reports helped us single out the most potent antigens among HCMV proteins: phosphoproteins pp150(UL32) and p52(UL44). Theoretical computer analysis of p52 epitopes showed the main antigenic determinants not cross-reacting with antigens of other viruses. Virus-containing (strain AD169) material was obtained and genome DNA was isolated. Amplification of a site of gene UL44 coding for unique determinants detected a PCR fragment of required electrophoretic mobility. The fragment was cloned in vector pLBE. The specificity of cloning was confirmed by restriction analysis of theoretical sites. Nucleotide sequence of cloned fragment of UL44 gene was studied by Maxam-Gilbert's method. Cloning in expressing bacterial vectors helped obtain HCMV recombinant protein p52 in the pure form and fused with beta-galactosidase. Enzyme immunoassay with HCMV-positive and negative donor sera and ABBOTT HCMV sera showed that recombinant p52 increased the sensitivity and specificity of a previously obtained recombinant pp150 as an antigen to HCMV-IgG and HCMV-IgM. The sensitivity and specificity is 100% with 98-99% reliability.
Subject(s)
Antigens, Viral/isolation & purification , Cytomegalovirus/immunology , DNA-Binding Proteins/isolation & purification , Viral Proteins/isolation & purification , Amino Acid Sequence , Antigens, Viral/chemistry , Antigens, Viral/genetics , Base Sequence , Cloning, Molecular , Cross Reactions , DNA, Viral , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Immunoenzyme Techniques , Molecular Sequence Data , Polymerase Chain Reaction , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
The genes encoding the precursors of secretory glycoprotein gp57-65 (A antigen) and glycoprotein complex gp100, gp60, gp48 (B antigen) of virulent strain JM of Marek's disease virus (MDV) have been cloned using PCR of viral DNA. Nucleotide sequences of both genes have been determined and amino acid sequence of the precursor peptides predicted, and compared with the corresponding sequences of other MDV strains. The results will be used for developing methods for the diagnosis of Marek's disease and vaccination against it.
Subject(s)
Antigens, Viral/genetics , Genes, Viral , Herpesvirus 2, Gallid/genetics , Viral Envelope Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Chickens , Cloning, Molecular , Herpesvirus 2, Gallid/immunology , Molecular Sequence DataABSTRACT
MT-4 cell line is a continuous strain of human T lymphocytes expressing defective noninfective subviral HTLV-1 particles. A fragment of sequence encoding the p24 protein and gene for envelope protein (env) have been obtained from genomic DNA of this culture by polymerase chain reaction. Both HTLV-1 fragments were cloned in bacterial vectors, and the nucleotide sequence of these regions was determined. The cloned DNA fragment encoding the p24 has only four point nucleotide exchanges. Analysis of the env gene structure revealed that the sequence had several amino acid exchanges and two deletions (13 bp and 70 bp).
Subject(s)
Human T-lymphotropic virus 1/genetics , Proviruses/genetics , Amino Acid Sequence , Bacteria/genetics , Base Sequence , Cell Line , DNA, Recombinant , Genes, env , Humans , Molecular Sequence Data , Point MutationABSTRACT
The epidemiological situation with cytomegalovirus infection in Siberia is still to be studied and serological diagnosis of human cytomegalovirus (HCMV) is not satisfactory. Two regions of HCMV genome (strain AD-169) have been examined for obtaining diagnostic reagents by expression cloning. Using polymerase chain reaction, fragments of gene of the immediate early protein (IE2) and of the region encoding for the entire hydrophilic part (1176 bp) of the large phospoprotein gene (pp150) have been obtained. Both fragments were cloned in bacterial vectors. Analysis of nucleotide sequence showed negligible substitutions in comparison with previously reported sequences for these genes.
