RESUMO
The anti-prion protein (PrP) monoclonal antibody T2 has previously been prepared using PrP-knockout mice immunized with mouse recombinant PrP residues 121-231, however its interaction mechanism to PrP antigen has not been cleared. Here we identified and characterized the epitope of T2 antibody. The competitive ELISA with 20-mer synthetic peptides derived from PrP121-231 showed that T2 antibody had no affinity for these peptides. The analysis with deletion mutants of PrP revealed that 10 amino acids in the N terminus and 66 amino acids in the C terminus of PrP121-231 were necessary for reactivity with T2. Two far regions are necessary for complete affinity of the T2 antibody for PrP; either region alone is not sufficient to retain the affinity. The epitope recognized by T2 antibody is discontinuous and conformational. We examined the effect of disulfide bond and salt bridges. Alkylation of cysteine residues in C terminus of PrP121-231, which breaks a disulfide bond and disrupts the structure, had diminished the reactivity. Mutations induced in the PrP121-231 to break the disulfide bond or salt bridges, markedly had reduced the reactivity with T2 antibody. It suggests that T2 antibody recognized the structure maintained by the disulfide bond and salt bridges.
Assuntos
Anticorpos Monoclonais , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Príons/química , Príons/imunologia , Alquilação , Animais , Reações Antígeno-Anticorpo , Sequência de Bases , Ligação Competitiva , Western Blotting , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/genética , Técnicas In Vitro , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Fragmentos de Peptídeos/genética , Príons/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
A bacterial strain isolated from the oral cavity of a healthy dog revealed an unusual colony formation in nebular appearance on agar plates. The isolated bacterial strain was Gram-positive, spore-forming rod with peritrichous flagella, and grown under aerobic conditions, but unable to grow at 45 degrees C. The strain was tentatively classified as Paenibacillus alvei according to the biochemical properties and the 16S rRNA gene sequence. The isolate exhibits collective locomotion on solid agar plates. The bacterial motility was inhibited with EDTA and was restored by adding magnesium. We concluded that magnesium ion is essential for collective locomotion of P. alvei. This suggests that EDTA is useful for inhibition of biofilm formation.
Assuntos
DNA Bacteriano/análise , Cães/microbiologia , Bactérias Gram-Positivas Formadoras de Endosporo/isolamento & purificação , Boca/microbiologia , RNA Ribossômico 16S/análise , Animais , Quelantes/farmacologia , Ácido Edético/farmacologia , Bactérias Gram-Positivas Formadoras de Endosporo/classificação , Bactérias Gram-Positivas Formadoras de Endosporo/genética , Magnésio/fisiologia , Dados de Sequência Molecular , RNA Ribossômico 16S/genéticaRESUMO
We have cloned the arginine deiminase (ADI) gene from Mycoplasma hominis PG21 genomic DNA by polymerase chain reaction, and changed four TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The recombinant ADI (rADI) was purified to apparent homogeneity by Ni-affinity chromatography after extraction from inclusion bodies followed by refolding. The rADI expressed in E. coli was estimated to be 50 kDa. Dimeric forms of rADI exerted enzymatic activity. We found that high concentration of potassium dihydrogenphosphate (PDP) and L-arginine addition in refolding reaction increases the enzyme activity. The specific activity of rADl was calculated as 0.618 U/mg. In addition, the enzyme activity of purified rADI remained for at least one month in 100 mM PDP solution (pH 6.5), but diminished within one week in 100 mM PDP solution (pH 7.4). Anti-tumor activity of the purified rADI was estimated to be 0.036 U/ml as 50% growth inhibitory activity against human melanoma cell line G-361.
Assuntos
Hidrolases/metabolismo , Hidrolases/farmacologia , Melanoma/metabolismo , Mycoplasma hominis/enzimologia , Proteínas Recombinantes , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Hidrolases/química , Hidrolases/genéticaRESUMO
Using a radiation-hybrid cell, E11, produced by the fusion of Hepa-1c1c7 and X-ray irradiated HepG2 cells, we located the omeprazole (OP) responsive region on chromosome 10p. The cDNA of 12 genes expressed in E11 cells were cloned from HepG2 mRNA by RT-PCR. The cDNA was transfected into Hepa-1c1c7 cells to check the increased expression of Cyp1a1 by reporter gene (luciferase) assay. Finally, one gene (tauCREM: cAMP responsive element modulator, tau-isoform) was identified as a candidate gene for the gene responsive to OP. The regulatory sequence of Cyp1a1 in response to tauCREM transfection was identified in one region (-60 to -52bp relative to the transcription start site), which was the basic transcription element (BTE). Electromobility shift assay with the BTE sequence showed an increase in the band intensity when the cells were treated with OP. Decreased level of endogenous CREM by siRNA transfection inhibited the induction of CYP1A1-mRNA in HepG2 cells by OP.
Assuntos
Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Receptores de Hidrocarboneto Arílico/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , DNA Complementar/metabolismo , Histonas/química , Humanos , Hibridização in Situ Fluorescente , Ligantes , Plasmídeos/metabolismo , Isoformas de Proteínas , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Hybridoma formation is an indispensable step in the production of monoclonal antibodies. Obtaining highly efficient fusion of an antibody-producing cell to the myeloma cell to form the hybridoma is an important step in this process. The electrofusion method is superior to chemical fusion methods such as the polyethylene glycol (PEG) method due to its high fusion efficiency. However, this method requires cell activation prior to electrofusion, a process that is time-consuming and tends to cause cell death. In this study, we achieved much higher fusion efficiency by stimulating B cells with CpG oligodeoxynucleotide (CpG ODN) over shorter periods. Splenocytes were isolated from immunized mice and cultured in the presence of a CpG ODN for 1 or 2 days. This CpG ODN stimulation evokes about one order of magnitude higher fusion efficiency than other stimulators. CpG ODN stimulation not only increases the fusion efficiency but also the number of antibody-producing cells. This leads to a substantial increase in the number of positive clones obtained. This highly efficient fusion method was used to produce a functional antibody against Gaussia luciferase. This method was found to produce greater numbers of hybridomas and to enable direct screening for antibodies with functional characteristics such as inhibition of the luminescence activity of an antigen. We were able to establish a functional antibody against Gaussia luciferase after a single fusion experiment using our electrofusion method.
Assuntos
Linfócitos B/efeitos dos fármacos , Fusão Celular , Hibridomas/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Copépodes/enzimologia , Ilhas de CpG/genética , Técnicas Eletroquímicas/métodos , Feminino , Hibridomas/imunologia , Hibridomas/metabolismo , Luciferases/imunologia , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/genética , Reprodutibilidade dos TestesRESUMO
We studied the role of the 2 salt bridges (Asp143-Arg147 and Asp146-Arg150) in helix 1 of mouse prion protein (PrP) on the formation of the complex between PrP and the monoclonal antibody T2. We introduced 6 charge-changing mutations to the amino acid residues associated with the salt bridges. Analysis of the circular dichroism spectra of the mutant PrPs showed that the salt bridge mutations did not change the secondary structures. We analyzed the kinetics of the association and dissociation of the PrPs with the T2 antibody. The results showed that the association kinetics were not significantly different among the variants except Arg150Lys, while the dissociation rate of the neutralized-charge variants was 2 orders of magnitude higher than that of the wild type. These results indicate that salt bridges make the interaction of PrP with T2 tighter by slowing down dissociation.