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Braz. j. med. biol. res ; 39(3): 345-354, Mar. 2006. ilus, tab
Article in English | LILACS | ID: lil-421367


Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vß genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable ß chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.

Animals , Female , Mice , Antigens, Protozoan/genetics , Genes, T-Cell Receptor/genetics , Glycoproteins/genetics , Immunodominant Epitopes/genetics , Interferon-gamma/genetics , Neuraminidase/genetics , Polymerase Chain Reaction/methods , Amino Acid Sequence , Antigens, Protozoan/immunology , Genes, T-Cell Receptor/immunology , Glycoproteins/immunology , Glycoproteins/metabolism , Hybridomas/metabolism , Immunodominant Epitopes/immunology , Interferon-gamma/immunology , Interferon-gamma , Mice, Inbred BALB C , Molecular Sequence Data , Neuraminidase/immunology , Neuraminidase/metabolism , Transcription, Genetic
Indian J Exp Biol ; 1998 Jan; 36(1): 70-5
Article in English | IMSEAR | ID: sea-62291


Variations in foot and mouth disease virus are due to amino acid substitutions in the VP1, which is a major immunogen. Analysis of this hypervariable region is essential to know the antigenic structure of the serotype and is necessary to select a suitable vaccine strain. FMDV type A22 is one of the four prevailing virus types for which the vaccine is used regularly. To understand the antigenic structure of this type, carboxy- terminal region of VP1 from two field isolates and vaccine virus were sequenced and analysed. The results indicate that, Indian A22 has distinct antigenic structure.

Amino Acid Sequence , Animals , Antigens, Viral/genetics , Aphthovirus/classification , Base Sequence , Capsid/genetics , Capsid Proteins , Cattle , DNA, Viral/genetics , Immunodominant Epitopes/genetics , Molecular Sequence Data , Sequence Homology, Amino Acid