RESUMO
An efficient and inexpensive laboratory approach for the generation and the purification of polyclonal antibodies to human antigen CD34 was developed. It was shown that cloned refolded and purified from Escherichia coli recombinant extracellular fragment of CD34 antigen retained immunogenic determinants of cell-surface expressed CD34. Immunization of mice with unglycosylated truncated recombinant protein elicit polyclonal antibodies specific for the native human antigen CD34. The antibodies generated are applicable for phenotyping of CD34+ cells using immunocytochemistry and flow cytometry assays.
Assuntos
Anticorpos/isolamento & purificação , Antígenos CD34/imunologia , Animais , Anticorpos/imunologia , Antígenos CD34/genética , Western Blotting , Linhagem Celular Tumoral , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Feminino , Vetores Genéticos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
A cDNA combinatorial antibody library of mouse variable immunoglobulin fragments has been constructed from mice immunized with rhIFN-beta1b. For this purpose, cDNAS of immunoglobulin variable heavy (V(H)) and variable light (V(L)) chains genes amplified from splenocytes were joined with linker DNA to form ScFv's (single-chain Fv-antibodies). The obtained ScFv-DNA pool was cloned into a phagemid vector and used for Esherichia coli transformation. Using the phage display technique, bacterial clones producing single-chain antibodies specific to rhIFN-beta1b were selected. The following characteristics of the combinatorial library were determined in this work: abundance, functional size, and the initial ScFv-DNA diversity in the library constructed. High specificity of interaction between phage displayed ScFv's and rhIFN-beta1b has been demonstrated.
Assuntos
Biblioteca Gênica , Fragmentos de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Interferon beta/imunologia , Animais , Clonagem Molecular , Técnicas de Química Combinatória , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Feminino , Humanos , Immunoblotting , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/imunologia , Interferon beta-1b , Camundongos , Camundongos Endogâmicos BALB CRESUMO
One of the most important problems in biotechnology today is development of simple and cheap techniques for protein purification. This is why inteins and protein splicing phenomena are of a great interest for protein purification. Modification of inteins by affinity tags permits to use general methods for protein purification. Following autocatalytic excision of tagged intein from protein allows to obtain pure protein without formyl-methionin residue on its N-terminus. New method for two step protein purification on the base of protein splicing phenomena has been created. Using modified intein Mxe GyrA extra pure human growth hormone with minimal lost and with native N-terminus was obtained.
Assuntos
Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/isolamento & purificação , Inteínas/genética , Processamento de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Biotecnologia/métodos , Hormônio do Crescimento Humano/biossíntese , Humanos , Proteínas Recombinantes de Fusão/biossínteseRESUMO
A panel of single-chain antibodies (ScFv's--single-chain Fv-antibodies) against recombinant human interferon beta 1b (rhIFN-beta1b) has been obtained from immune and naive combinatorial cDNA libraries of the mouse variable immunoglobulin genes. ScFv's were expressed into Escherichia coli cells. For producers isolated from the immune library a difference in production yield of ScFv's in periplasm and incubation medium as well as their expression stability in passages and storage stability have been demonstrated. After sequencing of target DNA the multiple alignment and structural analysis of ScFv's sequences with different primary structures were carried out and significant difference in both complementarity-determining (CDR) and framework (FR) regions of their variable domains has been shown. For the ScFv's isolated from the immune library, specificity of their binding with native and denatured rhIFN-beta1b in ELISA and Western-blotting as well as their high storage stability have been shown. The affinity constants for each representatives of the ScFv's panel were in the range from 1.96 x 10(-8) to 1.69 x 10(-9) M.
Assuntos
Anticorpos Monoclonais/imunologia , Fragmentos de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/imunologia , Interferon beta/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Western Blotting , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Biblioteca Gênica , Humanos , Immunoblotting , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Interferon beta-1b , CamundongosRESUMO
The insecticidal crystal proteins of 15 B. thuringiensis strains, isolated in the Crimea territory that are toxical for some Lepidoptera and Colorado potato beetle larvae were identified by PAGE electrophoresis. Ten strains produced the crystal proteins with high molecular weight (> 120 kD). PCR with use of broad specificity primers and DNA of these B. thuringiensis strains as template demonstrated the specific PCR products (1000 bp). Amplified DNA fragments were cloned and sequenced. The nucleotide sequence analysis revealed that the genomes of ten strains of B. thuringiensis carried Cry1B genes, which are responsible for production of the insecticidal crystal proteins with dual specificity. The influence of the solubilization conditions on the structure and toxicity of Cry1B protein for Colorado potato beetle larvae was shown. The dual toxicity of studied B. thuringiensis strains is explained by the Cry1B genes presence in their genomes. These strains may be used to develop the broad specificity bioinsecticides.
Assuntos
Bacillus thuringiensis/genética , Proteínas de Bactérias , Toxinas Bacterianas , Endotoxinas , Genes Bacterianos , Proteínas Hemolisinas , Microbiologia do Solo , Animais , Bacillus thuringiensis/isolamento & purificação , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/fisiologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacologia , Besouros/efeitos dos fármacos , Besouros/crescimento & desenvolvimento , DNA Bacteriano , Eletroforese em Gel de Poliacrilamida , Endotoxinas/genética , Endotoxinas/farmacologia , Endotoxinas/fisiologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Proteínas Hemolisinas/fisiologia , Larva/efeitos dos fármacos , Plasmídeos , UcrâniaRESUMO
The specific to 3 types of Cry genes primers containing inosine were constructed to find crystal insecticidal protein Bacillus thuringiensis genes using PCR. A number of new B. thuringiensis strains isolated in Ukraine were investigated using these PCR primers. As a results, Cry genes were found, some of them were identified and demonstrated high homology to Cry1Ba2 and Cry1Bc genes.
Assuntos
Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas , Primers do DNA/síntese química , Endotoxinas/genética , Genes Bacterianos/genética , Inosina , Inseticidas , Animais , Toxinas de Bacillus thuringiensis , Sequência de Bases , Besouros , DNA Bacteriano/genética , Proteínas Hemolisinas , Lepidópteros , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Homologia de Sequência do Ácido Nucleico , Microbiologia do Solo , UcrâniaRESUMO
The results obtained show the essential changes in functional state of hepatocyte's plasmatic membrane due to the implantation of human ApoA1 gene to the rat liver. The changes in phospholipid composition, hyperpolarization, increase in activity of membrane bound enzymes, cytochrome P-450 and biosynthesis of liver total proteins have been found. The essential changes characterizing cell effect were more marked in the adult rats, and membrane effect in the old ones.
Assuntos
Envelhecimento/patologia , Fígado/patologia , Transfecção , Animais , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Humanos , Fígado/enzimologia , Fígado/metabolismo , Lipídeos de Membrana/metabolismo , Potenciais da Membrana , Ratos , Ratos WistarAssuntos
Envelhecimento/fisiologia , Apolipoproteína A-I/genética , Técnicas de Transferência de Genes , Lipoproteínas HDL/fisiologia , Vasodilatação/fisiologia , Envelhecimento/sangue , Envelhecimento/genética , Animais , Aorta Torácica/fisiologia , Apolipoproteína A-I/sangue , Apolipoproteína A-I/química , Humanos , Técnicas In Vitro , Lipoproteínas HDL/sangue , Lipoproteínas HDL/química , Lipoproteínas HDL/genética , Músculo Liso Vascular/fisiologia , Ratos , Ratos Wistar , Vasodilatação/genéticaRESUMO
Evidences are given that implantation of human apo A-I gene to experimental animals induces essential changes in the functional state of hepatocyte plasmatic phospholipid composition, hyperpolarization development, as well as changes in the Na, K-ATPase and adenylate cyclase activities. The presence of generalized cell reaction is evidenced from the fact than biosynthesis of summary proteins gets enhanced during the above changes.
Assuntos
Apolipoproteína A-I/genética , Regulação da Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Fígado/metabolismo , Adenilil Ciclases/metabolismo , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Lipossomos , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de TempoRESUMO
Administration of laferon to patients stimulates the blood serum interferon formation, with its peak being reached by 24 h. The evaluation done confirmed the therapeutic efficacy of laferon in meningoencephalites. This was manifested by reduction in duration of certain clinical symptoms, timeterms of spinal fluid sanitation, improvement in immunity parameters and increase in the patients' blood serum level of interferon.
Assuntos
Infecções por Adenovirus Humanos/terapia , Antivirais/uso terapêutico , Infecções por Enterovirus/terapia , Infecções por Herpesviridae/terapia , Influenza Humana/terapia , Interferon Tipo I/uso terapêutico , Meningoencefalite/terapia , Infecções por Adenovirus Humanos/imunologia , Adulto , Avaliação de Medicamentos , Infecções por Enterovirus/imunologia , Feminino , Infecções por Herpesviridae/imunologia , Humanos , Imunidade Celular/imunologia , Influenza Humana/imunologia , Interferons/sangue , Interferons/efeitos dos fármacos , Masculino , Meningoencefalite/imunologia , Pessoa de Meia-Idade , Proteínas Recombinantes , Formação de Roseta , Fatores de TempoRESUMO
It is confirmed that inhibitors of DNA repair caffeine and 3-aminobenzamide decrease the survival of gamma-irradiated HeLa cells. It is shown that the decreased survival of irradiated cells is reversed when Escherichia coli RecA protein is introduced into cell nucleases with the aid of liposomes. This effect is more expressed in caffeine-treated (before or after irradiation) than in 3-aminobenzamide-treated (before irradiation) cells. It is suggested that E. coli 38 kD RecA protein may compensate the function of HeLa RecA-like protein, inhibited by DNA repair inhibitors, which is necessary for the repair of single-strand breaks and double-strand breaks of DNA.
Assuntos
Benzamidas/farmacologia , Cafeína/farmacologia , Reparo do DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Recombinases Rec A/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Portadores de Fármacos , Interações Medicamentosas , Raios gama , Células HeLa/efeitos da radiação , Humanos , Lipossomos , Recombinases Rec A/administração & dosagem , Recombinases Rec A/isolamento & purificaçãoAssuntos
Envelhecimento/genética , Apolipoproteínas A/genética , Lipoproteínas HDL/genética , Transfecção/genética , Envelhecimento/sangue , Animais , Apolipoproteína A-I , Apolipoproteínas A/sangue , Clonagem Molecular , DNA/genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Lipoproteínas HDL/sangue , Fígado , Plasmídeos/genética , RatosRESUMO
Enzyme immunoassays for the detection of viral antigens and virus-specific antibodies in biological samples have been described. Molecular complexes of antibodies and beta-lactamase (penicillinase) have been used as anti-specific conjugates. To synthesize the conjugate, the enzyme obtained with the aid of genetic engineering has been used. Enzyme immunoassays have been tested for the indication of the influenza virus and virus-induced specific antibodies. Enzyme immunoassays were shown to possess certain advantages (e.g., the use of simple and nontoxic substrate) along with the sensitivity identical to that of other methods, employing peroxidase-based conjugates.
Assuntos
Anticorpos Antivirais/análise , Especificidade de Anticorpos , Técnicas Imunoenzimáticas , Vírus da Influenza A/isolamento & purificação , Estudos de Avaliação como Assunto , Engenharia Genética , Vírus da Influenza A/imunologia , beta-LactamasesAssuntos
Escherichia coli/genética , Regulação Fúngica da Expressão Gênica , Genes Bacterianos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transformação Genética , beta-Lactamases/genética , DNA Bacteriano/genética , Escherichia coli/enzimologia , Mutação , Plasmídeos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , beta-Lactamases/análiseRESUMO
It is shown that a degradation level of exogenic high-polymeric RNA does not change their stimulating effect on beta-galactosidase synthesis in yeast protoplasts. Nucleoside monophosphates are a product of RNA degradation which promotes an increase in the beta-galactosidase activity. The exogenic RNA is established to act at the transcription level. RNA and its degradation products stimulate the beta-galactosidase synthesis without any effect on the intensity of the total RNA and protein synthesis in the yeast protoplasts. All mentioned permitted a conclusion that products of RNA decay participate in the regulation of the beta-galactosidase synthesis of yeast. A possible mechanism of their action is under discussion.
Assuntos
Galactosidases/metabolismo , RNA/farmacologia , Saccharomyces/enzimologia , beta-Galactosidase/metabolismo , Hidrólise , Oligorribonucleotídeos/farmacologia , Oxiquinolina/farmacologia , RNA Fúngico/biossíntese , Saccharomyces/metabolismoRESUMO
Expression of Ap and Tc genes of the plasmid origin in lambda vectors where the effect of lac promoter and PL phage promoter can be traced has been studied. The different orientation of Ap gene relative to strong promoters (lac and PL) permitted the beneficial arrangement of Ap gene for its optimum expression to be established. The interfering interaction of the two closely arranged strong promoters, Ap gene and lac promoter, has been detected. This repressed considerably the expression of Ap gene. The enhanced expression of this gene has been observed under the effect of the removed PL phage promoter. The active expression of Tc gene has been only observed in clones with the recombinant lambda-pcv-20 molecule (without lac promoter), irrespective of the plasmid pcv-20 orientation. This, presumably, can be accounted for by the favourable secondary structure of the m-RNA of Tc gene providing the efficient translation.
Assuntos
Bacteriófago lambda/genética , Regulação da Expressão Gênica , Genes Bacterianos , Vetores Genéticos , Plasmídeos , Mapeamento Cromossômico , Escherichia coli/genética , Óperon , Recombinação Genética , TransfecçãoRESUMO
The method for obtaining nonlysogenic bacteria from lysogenic ones by means of a phage carrying resistance to antibiotics is proposed. Solitary nonlysogenic cells in a lysogenic culture are lysogenized after the infection of the culture with a labeled phage and then harvested on a selective medium: under special conditions the phage is eliminated from the cells.
