Expression of cloned envelope protein genes from the flavivirus tick-borne encephalitis virus in mammalian cells and random mutagenesis by PCR
Identifieur interne : 001A86 ( Istex/Checkpoint ); précédent : 001A85; suivant : 001A87Expression of cloned envelope protein genes from the flavivirus tick-borne encephalitis virus in mammalian cells and random mutagenesis by PCR
Auteurs : Steven L. Allison [Autriche] ; Christian W. Mandl [Autriche] ; Christian Kunz [Autriche] ; Franz X. Heinz [Autriche]Source :
- Virus Genes [ 0920-8569 ] ; 1994-03-01.
English descriptors
- KwdEn :
- Teeft :
- Amino, Amino acid changes, Amino acid residues, Amino acid sequence, Amino acids, Amplification, Amplitaq polymerase, Antigenic domains, Antigenic structure, Appropriate context, Base pair, Capsid protein, Cdna, Cdna synthesis, Cells transfected, Cfrloi, Clone, Clones encoding, Codon, Error frequency, Flavivirus, Functional activities, Further increase, Gene, Guirakhoo, Heinz, Host signalase, Ideal rate, Immature virions, Immunofluorescence, Immunofluorescence assay, Indirect immunofluorescence, Intracellular, Intracellular level, Japanese encephalitis virus, Kunz, Limited number, Lysis buffer, Mabs, Mammalian cells, Mandl, Mason virology, Membrane proteins, Methods applic, Molecular interactions, Monoclonal antibodies, Mutagenesis, Mutation, Natural error frequency, Noti, Nucleotide, Oligonucleotide primers, Perkin elmer cetus, Plasmid, Plasmid clones, Plasmid expression vectors, Plasmid vector, Polymerase, Polymerase chain reaction, Potential value, Present study, Primer, Protein, Random mutagenesis, Random mutations, Recombinant plasmids, Region series, Restriction enzyme noti, Restriction enzymes, Room temperature, Secretory pathway, Sequencing reactions, Silent mutation, Specific regions, Standard conditions, Strong fluorescence, Structural model, Structural proteins, Transfected, Transfection, Viral, Viral assembly, Viral protease, Virology, Virus, Virus antigen, Virus gene expression, Virus pretreated.
Abstract
Abstract: The structural membrane proteins prM and E of the flavivirus tick-borne encephalitis (TBE) virus were expressed in mammalian cells for the purpose of probing the structure and molecular interactions of these proteins. Advantage was taken of the natural error frequency of the Taq polymerase used in the PCR amplification to generate a randomly mutated population of genes that were then cloned directly into plasmid expression vectors under the control of an SV40 promoter. Analysis of the mutation frequency by direct sequencing of 22 separate clones showed that the PCR produced mutations at a rate yielding an average of one to two amino acid changes per clone in the 496 amino acid long protein E. This is an ideal rate for assessing the importance of individual amino acid residues within protein domains, thus demonstrating the potential value of the PCR as a random mutagenesis method. Clones encoding wild-type prM and E proteins, and a truncated form of E, were also constructed by recombining portions of selected PCR clones. Transfection of COS-1 cells with these constructs resulted in expression of the prM and E proteins, which was demonstrated by indirect immunofluorescence using monoclonal antibodies (Mabs). The intracellular level of TBE virus antigen, measured in lysates of transfected cells by ELISA, reached approximately 25% of that found in virusinfected COS cells. Furthermore, it was shown by immunofluoresence using a panel of 19 anti-E Mabs that the antigenic structure of the expressed E proteins was nearly identical to that of E protein in infected cells, thus confirming the suitability of this model system as a tool for studying flavivirus protein structure.
Url:
DOI: 10.1007/BF01703077
Affiliations:
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ISTEX:473CA05457BE44D507CA0756ED01BF625FC4427DLe document en format XML
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<term>Amino acid residues</term>
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<term>Amino acids</term>
<term>Amplification</term>
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<term>Immature virions</term>
<term>Immunofluorescence</term>
<term>Immunofluorescence assay</term>
<term>Indirect immunofluorescence</term>
<term>Intracellular</term>
<term>Intracellular level</term>
<term>Japanese encephalitis virus</term>
<term>Kunz</term>
<term>Limited number</term>
<term>Lysis buffer</term>
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<term>Polymerase chain reaction</term>
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<term>Sequencing reactions</term>
<term>Silent mutation</term>
<term>Specific regions</term>
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<term>Strong fluorescence</term>
<term>Structural model</term>
<term>Structural proteins</term>
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<term>Transfection</term>
<term>Viral</term>
<term>Viral assembly</term>
<term>Viral protease</term>
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<front><div type="abstract" xml:lang="en">Abstract: The structural membrane proteins prM and E of the flavivirus tick-borne encephalitis (TBE) virus were expressed in mammalian cells for the purpose of probing the structure and molecular interactions of these proteins. Advantage was taken of the natural error frequency of the Taq polymerase used in the PCR amplification to generate a randomly mutated population of genes that were then cloned directly into plasmid expression vectors under the control of an SV40 promoter. Analysis of the mutation frequency by direct sequencing of 22 separate clones showed that the PCR produced mutations at a rate yielding an average of one to two amino acid changes per clone in the 496 amino acid long protein E. This is an ideal rate for assessing the importance of individual amino acid residues within protein domains, thus demonstrating the potential value of the PCR as a random mutagenesis method. Clones encoding wild-type prM and E proteins, and a truncated form of E, were also constructed by recombining portions of selected PCR clones. Transfection of COS-1 cells with these constructs resulted in expression of the prM and E proteins, which was demonstrated by indirect immunofluorescence using monoclonal antibodies (Mabs). The intracellular level of TBE virus antigen, measured in lysates of transfected cells by ELISA, reached approximately 25% of that found in virusinfected COS cells. Furthermore, it was shown by immunofluoresence using a panel of 19 anti-E Mabs that the antigenic structure of the expressed E proteins was nearly identical to that of E protein in infected cells, thus confirming the suitability of this model system as a tool for studying flavivirus protein structure.</div>
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