[Synthesis of chimeric IgE in vivo after ballistic transfection of immunoglobulin genes in various mouse tissues].
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Animals↗
Biomedical subjects
S M Deev
Publications and source records attributed to S M Deev.
[Modified ribonuclease gene provides efficient positive selection in molecular cloning].
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Base Sequence↗
[Biosynthesis of mini-antibodies to human ferritin in plant and bacterial producers].
A recombinant scFv antibody against human spleen ferritin was expressed as a barstar-fused protein in Escherichia coli and in Nicotiana tabacum plants and suspension cell cultures. As demonstrated by immunoblotting with antibarstar antibodies, direction of the recombinant protein to the endomembrane system of plant cells ensured its stability and solubility. Production of the recombinant protein did not differ between parental transgenic plants and their first-generation progeny. Fusion with barstar allowed not only immunochemical detection of the recombinant scFv antibody, but also their purification from the plant material by affinity chromatography with barnase-His6 immobilized on a metal-affinity carrier.
Bacteria↗
[Thermostability of alkaline phosphatase of Meithermus ruber bacteria: cloning of the gene, expression in Escherichia coli cells and biochemical characterization of the recombinant protein].
The Meiothermus ruber alkaline phosphatase gene was cloned, expressed in Escherichia coli cells, and sequenced. The enzyme precursor, including the putative signal peptide, was shown to consist of 503 residues (deduced molecular mass 54,229 Da). The recombinant enzyme showed the maximal activity at 60-65 degrees C and pH 11.0 and had K(m) = 0.055 mM as estimated with p-nitrophenyl phosphate (pNPP). The enzyme proved to be moderately thermostable, retaining 50% activity after 6 h incubation at 60 degrees C and being completely inactivated in 2 h at 80 degrees C. In substrate specificity assays, the highest enzymic activity was observed with pNPP and dATP. Vanadate, inorganic phosphate, and SDS inhibited M. ruber alkaline phosphatase, while thiol-reducing agents had virtually no effect. The enzymic activity strongly depended on exogenous Mg2+ and declined in the presence of EDTA.
Alkaline Phosphatase↗
[The antiviral activity of RNAse Bacillus intermedius in experiments with mice preinfected with street rabies virus].
RNAse Bacillus intermedius, when administered once and according to 11 repeated experiments, protected the preliminarily infected CBA mice with street rabies virus (protection of 40-67%; p < 0.01-0.001). A reliable protection of Animals was registered only when RNAse was administered intramuscularly at the virus introduction spot; it was not effective, when the bacterial RNAse was injected in the brain, vein, under the skin or in muscles of a non-infected extremity. Neither did it produce any suppressive effect on the vaccinal antirabic immunity.
Animals↗
[Antiviral activity of Bacillus intermedius RNAase in guinea-pigs and rabbits infected with outdoor rabies virus].
Single injection of Bacillus intermedius RNAase in a dose of 5 mg/kg could protect 40 and 50-70% of the outdoor rabies virus-preinfected guinea-pigs and rabbits, respectively. In the control group there were 100 and 75-100% deaths of the RNAase-untreated guinea-pigs and rabbits, respectively. Animal protection was observed only when RNAase was injected into the site of viral administration. The intramuscular injection of RNAase, other than the site of viral administration failed to protect the infected animals. The efficacy (75%) of RNAase injected into the rabbits was similar 1 and 24 hours after animal infection.
Animals↗
[Immunoglobulin genes in lymphoid cells and regulation of their transcription].
The hybridoma genomes contain polyploid sets of immunoglobulin genes. We have shown, that the hybridoma PTF-02 genome contains three genes of heavy chains and two genes of light chains. The genes responsible for antibody synthesis were cloned and their structure were determined. Investigation of the kappa gene transcription and its fragments which contain regulatory sequences revealed a nuclear factor. The latter interacts with the octanucleotide localized at the promoter region of the kappa gene. The purified factor activates the transcription of the kappa gene in a heterologous cell-free system. Together with the tissue-specific factor there is also an universal factor interacting with the octanucleotide sequence. We have shown an additional factor in lymphoid cells interact with the protein which binds to the octanucleotide sequence. We have shown an additional factor in lymphoid cells interacting with the protein which binds to the octanucleotide sequence. As a result, there is a family of factors which interact with ATTTGCAT sequence. One major factor (m.w. 60 +/- 2 kDa) is an obligatory component for the initiation of immunoglobulin genes transcription.
Animals↗
[Immunoglobulin kappa-genes. Cloning, hybridization and structural analysis].
The principal problems in molecular and genetic immunology to be resolved are the structure of Ig-genes and the regulation of their expression. The isolation of mRNA for light and heavy Ig-chains would be a first step along this line. A combination of two approaches may be the best strategy in mRNA preparation. Affinity purification allows one to obtain pure mRNA in a one-step procedure whereas immunoprecipitation makes it possible to prepare mRNAs for both heavy and light immunoglobulin chains in considerable amounts. In the series of experiments, conditions for synthesis of long and short cDNA chains were elaborated and the clone containing the fragment of kappa-chain gene was isolated and characterized. This clone was used as a probe for hybridization with genomic DNA from myeloma and hybridoma cells. It was shown that both allelic genes on homologous chromosomes were rearranged in myeloma MOPC21 cells. The original cell line of hybridoma PTF02 contains the embryonic gene as well as the differentiated genes. However, only differentiated genes can be detected in a similar experiment conducted with the same hybridoma after passage on mice. In conclusion, the coordination of homologous and heterologous chromosome expression in B cells in discussed in terms of the feed-back control.
Amino Acid Sequence↗
[Synthesis of double-stranded DNA on light immunoglobulin chain matrix RNA].
cDNA was synthesized on Ig kappa-L-chain mRNA isolated from mouse plasmocytoma MOPC 21 using avial myeloblastosis RNA-dependent DNA polymerase. The cDNA was used as a matrix for second DNA chain synthesis using 5'-exonuclease free DNA-polymerase I (Klenoff fragment). Hairpin DNA having double length of initial cDNA was obtained without added exogenous primer. SI nuclease treatment of the hairpin DNA results in reducing the DNA length twice, as shown by electrophoresis in denaturing conditions. The fact, that S1 nuclease resistance of the hairpin DNA is 75% shows high complementarity between first and second DNA chain. The product length obtained after S1 nuclease treatment was shown to be heterogenous. The maximal length of the product, reaching at least 900 base pairs, is near to be initial mRNA length (without the poly(A)-fragment).
Animals↗
[Group-selective immunoanalysis].
The new solid-phase immunoassay technique has been applied for antidigoxin monoclonal antibodies (Mabs) detection. At the first assay step, antidigoxin Mabs (IgG1, Kaff = 9.2 x 10(9) M-1) bound with specially prepared immunosorbent: microtiter plates with adsorbed digoxin-human serum albumin conjugate, in which free amino groups were blocked by glutaraldehyde (the microtiter plates with immobilized conjugate were treated for 3h by 2.5% glutaraldehyde at 45 degrees C, this had no significant effect on the immunological properties of immunosorbents). After antigen-antibody reaction and solid phase separation, free amino groups were located only at the antidigoxin Mabs bound to GSI immunosorbent. At the second assay step, N-hydroxysuccinimide-biotin ester solution was added to bind with the free amino groups. After separation at the third assay step, biotin labels were detected by the streptavidin-peroxidase conjugate. The method does not require second labeled antibodies and may be used for anti-hapten screening.
Antibodies, Monoclonal↗
[Expression of "chimeric" (murine/human) immunoglobulin genes in lymphoid and nonlymphoid cells].
A recombinant tandem of mouse/human immunoglobulin (Ig) genes was constructed and inserted in the plasmid pGEM1 under the control of the T7 RNA polymerase promoter. Constant Ig genes of murine origin were replaced with human genomic ones. Isotype IgG1 was changed for IgE. Lymphoid cell line Sp2/0 and CHO (chinese hamster ovary) cells were used for transfection. Both cell lines contained the semisynthetic gene of T7 RNA polymerase in their genomes and stably expressed this polymerase. Transfected clones of Sp2/0 and CHO cells synthesized k-light (25 kD) and epsilon-heavy (70 kD) chains of Ig and the corresponding mRNAs. Both lymphoid and nonlymphoid transfected cell lines synthesized functionally active antibodies interacting with the antigen (pig transferrin). Particular clones synthesized up to 150 ng/ml of chimeric antibodies. However, only lymphoid Sp2/0 cells were capable of efficient secretion. Nonlymphoid CHO cells were able to produce antibodies, but incapable of their efficient secretion.
Animals↗
[Functioning and silent regrouped immunoglobulin genes in the hybridoma genome].
Rearranged genes for heavy and light immunoglobulin chains have been studied in the genomes of hybridoma PTF-02 and parent myeloma P3-X63.Ag8.653. The hybridoma was shown to contain three rearranged allelic variants of the heavy chain gene's locus. Gene H2, responsible for the synthesis of the heavy chain of the antibody to transferrin, was transmitted to the hybridoma cell from a lymphocyte. The structure of the 5'-terminal codons of this gene corresponds to N-terminal amino acid residues of a heavy chain expressed by PTF-02 hybridoma. Two other genes (H1 and H3) were found both in the hybridoma and parent myeloma. Both H1 and H3 genes have serious defects in their structure and do not function. Rearranged k-genes were also identified both in the hybridoma and parent myeloma. A functional (K2) gene appeared in the hybridoma genome from an antigen-stimulated lymphocyte. The fetal mouse Vk gene, which has high homology (96.5%) with the K2 gene, was cloned and sequenced. Both K2 and the Vk fetal gene belong to one subfamily of k-genes. The rearranged gene K1 is nonfunctional because it has lost the Jk locus. This gene was transmitted from myeloma used for fusion. In such a way, the origin of all rearranged heavy and light chain genes in hybridoma PTF-02 was established.
Alleles↗
[Preparation of single-chained antibodies to human ferritin in Escherichia coli].
We have cloned cDNA copies of the immunoglobulin heavy and light chain genes from hybridoma cells able to produce antibody against human ferritin. Variable segments of these genes were obtained using the polymerase chain reaction (PCR). The specific amplifications of ligase reaction products were carried out to combine the variable segments with DNA fragments coding for a peptide-linker and for a signal peptide of the cloned pelB gene of Erwinia carotovora. During the antiferritin single-chain antibody gene expression under the T7 RNA polymerase control in E. coli the processed molecules of recombinant proteins formed aggregates in periplasm. The reversible denaturation in the absence of reducing agents had allowed us to obtain single-chain antibodies with the original binding specificity toward human ferritin.
Amino Acid Sequence↗