ABSTRACT
The lysate and recombinant antigens of various production included informula of ELISA-test-systems were analyzed. The ELISA-test-systems are used for detection of IgG to Herpes simplex virus type I and II. For testing the panel of serums PTH 201 (BBI Inc.) were used. The samples of this panel contain antibodies to Herpes simplex virus type I and II in mixed titers. The 69 serums of donors were used too (17 samples had IgG to Herpes simplex virus type I, 23 samples to Herpes simplex virus type II and 29 samples had no antibodies to Herpes simplex virus). The diagnostic capacity of mixture of recombinant antigens gG1 Herpes simplex virus type I and gG2 Herpes simplex virus type II (The research-and-production complex "DiaprofMed") was comparable with mixture of lysate antigen Herpes simplex virus type I and II (Membrane) EIE Antigen ("Virion Ltd."). In the test-systems for differentiation of IgG to Herpes simplex virus type I the recombinant antigen gG1 Herpes simplex virus type I proved to be comparable with commercial analogue Herpes simplex virus-1 gG1M ("Viral Therapeutics Inc."'). At the same time, capacity to detect IgG to Herpes simplex virus type II in recombinant protein gG2 Herpes simplex virus type II is significantly higher than in its analogue Herpes simplex virus-2 gG2c ("Viral Therapeutics Inc.").
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Herpes Simplex/diagnosis , Herpesvirus 1, Human/isolation & purification , Herpesvirus 2, Human/isolation & purification , Antigens, Viral/blood , Antigens, Viral/immunology , Antigens, Viral/isolation & purification , Herpes Simplex/immunology , Herpes Simplex/virology , Herpesvirus 1, Human/pathogenicity , Herpesvirus 2, Human/immunology , Herpesvirus 2, Human/pathogenicity , Humans , Immunoglobulin G/blood , Recombinant Proteins/bloodABSTRACT
To reveal Herpes simplex virus 2 specific IgG and to determine their avidity the ELISA test-kit was constructed using recombinant protein gG2 (PSC SPC Diaproph-Med). Using this test-kit the distribution of specific antibodies with different avidity indexes was investigated in practically healthy donor samples. It is possible to use the mentioned ELISA test-kit for confirmation of primary infection, and also for differentiation of primary infection, chronic disease and virus reactivation. Thus, this ELISA test-kit could be an additional tool in herpetic infection diagnosis.
Subject(s)
Antibodies, Viral , Antibody Affinity/immunology , Herpes Genitalis/diagnosis , Herpesvirus 2, Human/physiology , Immunoglobulin G , Antibodies, Viral/immunology , Chronic Disease , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Herpes Genitalis/immunology , Herpes Genitalis/virology , Humans , Immunoglobulin G/immunology , Plasmids , Reagent Kits, Diagnostic , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Severity of Illness Index , Virus ActivationABSTRACT
It is shown that the recombinant protein GST-HSV2gG, containing the immunodominant regions of glycoprotein G of HSV-2 is accumulated in the form of inclusion bodies or in soluble form in the Escherichia coli BL21 (DE3) cells. The ratio between protein fractions varied depending on the physiological state of cells before biosynthesis. The kinetic parameters of bacterial populations were determined by mathematical modeling of growth curves based on the Verhulst logistic function. It was established that the induction of biosynthesis in the growth acceleration phase (at OD600 = 0.3) with 0.1 mM IPTG gives the maximum yield of soluble protein (26.75 mg/l or 17.6 mg/g biomass). The target protein was purified using the immobilized metal ion affinity and affinity chromatography technologies. Antigenic activity of the soluble form of recombinant protein GST-HSV2gG, was significantly (three times) higher than that of the protein purified from inclusion bodies (p < 0.05) and was comparable with the activity of the commercial analog (p > 0.05), that allows using this product in the immunosorbent test kits for diagnosis of IgG to HSV-2.
Subject(s)
Antigens/biosynthesis , Escherichia coli/physiology , Recombinant Fusion Proteins/biosynthesis , Viral Envelope Proteins/biosynthesis , Antigens/genetics , Antigens/isolation & purification , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/growth & development , Isopropyl Thiogalactoside/pharmacology , Models, Biological , Plasmids , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Solubility , Viral Envelope Proteins/genetics , Viral Envelope Proteins/isolation & purificationABSTRACT
AIM: To evaluate expression patterns of protein product of putative tumor suppressor gene TSC-22 in human astrocytic tumors by immunohistochemical approach. METHODS: Plasmid pET-23d-TSC22 was constructed for the expression of human TSC-22 protein in bacterial system, and polyclonal rabbit antibodies against recombinant TSC-22 were produced. Immunohistochemical analysis of TSC-22 and GFAP expression with the use of anti-human-TSC-22- and anti-human-GFAP-antibodies was performed on histological slides of astrocytic tumors. RESULTS: Immunohistochemical analysis has shown that the number of cells expressing TSC-22 was significantly lower in glioblastoma tissues than that in diffuse astrocytoma. Double immunohistochemical staining of astrocytic tumors using anti-human-TSC-2- and anti-human-GFAP-antibodies showed that both TSC-22 and GFAP expression is co-localized in astrocytes. CONCLUSION: TSC-22 protein is expressed in astrocytes, but not in macrophage/microglial cells. In more aggressive forms of astrocytic tumors decreased expression of TSC-22 mRNA correlates with its lowered expression on protein level.
Subject(s)
Astrocytoma/metabolism , Brain Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Repressor Proteins/biosynthesis , Amino Acid Sequence , Astrocytes/metabolism , Astrocytoma/pathology , Base Sequence , Biomarkers, Tumor/analysis , Brain Neoplasms/pathology , Gene Expression Profiling , Glial Fibrillary Acidic Protein/biosynthesis , Humans , Immunohistochemistry , Microglia/metabolism , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Repressor Proteins/geneticsABSTRACT
A recombinant phage mp9MW/rpoB containing the BglII-B fragment of the Escherichia coli rplJL-rpoBC gene cluster was constructed on the basis of filamentous phage M13. Stability of the phage was increased by insertion of a transcription terminator t beta' which blocked transcription of cloned genes from the Plac of the vector.
Subject(s)
Coliphages/genetics , Escherichia coli/genetics , Genes, Bacterial , Recombination, Genetic , Cloning, Molecular , Gene Expression Regulation, Viral , Genes, Viral , PlasmidsABSTRACT
E. coli DNA fragment containing the rpoB gene with an rpoB3 rifampicin resistance dominant mutation (coding for the beta-subunit of RNA polymerase), genes rpI J and rpI L coding for the ribosomal proteins L7/L12 and L10, and promoters determining transcription of all these genes were cloned in M13mp8 and WB2348 filamentous phages. E. coli cells containing recombinant phages acquired resistance to rifampicin up to its 600 micrograms/ml concentration. When cloned into M13mp8 and WB2348 phages, the given fragment is oriented in such a way that the direction of the transcription initiated from its own promoter coincides with that initiated from the lac UV5 promoter. In both cases the recombinant phages have no stable rifampicin resistance which is coded by the fragment.
