RESUMO
The adenomatous polyposis coli (APC) tumor suppressor protein is involved in the Wnt/wingless pathway, modulating beta-catenin activity. We report the development of a highly specific, chemically synthesized oligobody (oligonucleotide-based synthetic antibody), directed against the N-terminal region of APC. Using this reagent, we found that within 16 h of disrupting HT-29 cell-cell contacts by harvesting cells with trypsin/EDTA treatment and replating, APC was translocated from the cytoplasm to the nucleus. Five days after plating the cells, when the cells had returned to their normal confluent phenotype and cell-cell contacts were reestablished, APC returned to the cytoplasm. These results suggest that APC functions as part of a "sensor" system, and responds to the loss of cell-cell contacts by moving to the nucleus, and returning to the cytoplasm when the contacts are fully restored.
Assuntos
Comunicação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Genes APC , Proteína da Polipose Adenomatosa do Colo , Anticorpos , Anticorpos Monoclonais , Núcleo Celular/metabolismo , Neoplasias do Colo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/genética , Biblioteca Gênica , Humanos , Junções Intercelulares/fisiologia , Microscopia Confocal , Oligodesoxirribonucleotídeos/química , Transporte Proteico , Moldes Genéticos , Células Tumorais CultivadasRESUMO
Oligonucleotide aptamer(s), obtained by using the SELEX procedure, has been used as reagents to recognize different molecules with high affinity and specificity. However, until recently, it was not possible to obtain oligonucleotide-based reagents able to recognize proteins with high specificity in assays typical of antibodies, such as immunohistochemistry, Western blotting and immunoprecipitations. Here, we show the results obtained by applying the strategy of "target switching" to obtain specific polyclonal and monoclonal oligobodies against the protein ERK2. We were able to develop highly specific polyclonal oligobodies by using only one selection step with a temporary target and one selection step with the final target (ERK2). Since only two selection steps were required, these results demonstrate that it is possible to obtain specific reagents against a protein without a need for an "in vitro evolution" using many selection steps, or error-prone polymerases. After one additional selection step, the polyclonal oligobodies were cloned to obtain a highly specific monoclonal oligobody.
Assuntos
Anticorpos Monoclonais/imunologia , Proteína Quinase 1 Ativada por Mitógeno/análise , Sequência de Aminoácidos , DNA de Cadeia Simples/imunologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Dados de Sequência Molecular , Desnaturação Proteica , Células Tumorais CultivadasRESUMO
Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first "synthetic antibody" reported.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Oligonucleotídeos/síntese química , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Imuno-Histoquímica , Camundongos , Oligonucleotídeos/imunologia , Oligonucleotídeos/isolamento & purificação , Reação em Cadeia da Polimerase , Testes de Precipitina , CoelhosRESUMO
Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first [quot ]synthetic antibody[quot ] reported.
RESUMO
Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first [quot ]synthetic antibody[quot ] reported.
Assuntos
Animais , Coelhos , Ratos , Oligonucleotídeos/síntese química , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Oligonucleotídeos/imunologia , Testes de Precipitina , Imuno-Histoquímica , Western Blotting , Reação em Cadeia da Polimerase , Anticorpos Monoclonais/imunologiaRESUMO
Using synthetic peptides and a combinatorial library of 56 mer random oligonucleotides, we have developed reagents that behave as "synthetic antibodies". The results obtained with the protein phosphatase 2A as a model system are shown here. The specificity of these reagents, named "oligobodies", has been demonstrated by Western blot analysis and immunohistochemistry. The oligobodies have enormous advantages compared to antibodies: their production is independent of the immune system, they can be prepared in a few days and there is no need for a purified target protein. These reagents can be produced even if the corresponding protein was never isolated or purified, since only a partial DNA sequence from a database provides enough information to make them.
Assuntos
Especificidade de Anticorpos , Oligonucleotídeos/síntese química , Biblioteca de Peptídeos , Animais , Western Blotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos/imunologia , Fosfoproteínas Fosfatases/imunologia , Reação em Cadeia da Polimerase , Proteína Fosfatase 2 , Coelhos , Análise de Sequência de DNARESUMO
Using synthetic peptides and a combinatorial library of 56 mer random oligonucleotides, we have developed reagents that behave as [quot ]synthetic antibodies[quot ]. The results obtained with the protein phosphatase 2A as a model system are shown here. The specificity of these reagents, named [quot ]oligobodies[quot ], has been demonstrated by Western blot analysis and immunohistochemistry. The oligobodies have enormous advantages compared to antibodies: their production is independent of the immune system, they can be prepared in a few days and there is no need for a purified target protein. These reagents can be produced even if the corresponding protein was never isolated or purified, since only a partial DNA sequence from a database provides enough information to make them.
RESUMO
The association of HLA antigens and type I or "lupoid" CAH-C was investigated in a population of 52 Argentinian Caucasoid patients. When compared with a population of normal individuals of the same ethnic group (n = 197), a significant increase of HLA-DR6 was observed (68.6% in patients vs 17.3% in controls; RR = 12.3, chi 2 = 52.4, pc = 0.00001). DNA typing showed that the HLA-DRB1*1301 allele was present in 32 out of 33 HLA-DR6 patients (66.6% of all the C-CAH patients vs 10.5% in controls; RR = 16.2, chi 2 = 111.3, pc = 0.00001). Analysis of HLA-DQB1 alleles also showed a significant increase of DQB1*0603 (RR = 15.4, chi 2 = 106.5, pc = 0.00001), an allele found in strong linkage disequilibrium with DRB1*1301. The association of CAH-C with this particular HLA-DR6 haplotype has not been previously described for the adult onset CAH. This different HLA predisposition, together with the fact that extrahepatic autoimmune diseases occur frequently only in the adult form of the disease, suggest that the immunopathogenic mechanisms involved in the development of these diseases may be different.