Expression in Escherichia coli of two Schistosoma mansoni genes that encode major antigens recognized by immune mice
Identifieur interne : 001928 ( Istex/Corpus ); précédent : 001927; suivant : 001929Expression in Escherichia coli of two Schistosoma mansoni genes that encode major antigens recognized by immune mice
Auteurs : David E. Lanar ; Edward J. Pearce ; Alan SherSource :
- Molecular & Biochemical Parasitology [ 0166-6851 ] ; 1985.
Abstract
Two clones which contain genes encoding Schistosoma mansoni proteins recognized by immune mouse sera were chosen from a cDNA λgt11 expression vector library by preselecting clones from the library with rabbit antisera against adult worm phosphate-buffered saline (PBS)-soluble antigens. One clone, MAC 182, codes for part of a Mr 70 000 protein; the other clone, MAC 184, codes for a Mr 27 000 protein. The insert sizes of MAC 182 and MAC 184 are 400 bp and 800 bp, respectively. Both clones express S. mansoni β-galactosidase fusion proteins as products of the construct. Antibodies from either chronically infected mice or mice vaccinated with irradiated cercariae recognize the MAC 182 fusion protein (MAC 182fp) but not the MAC 184 fusion protein (MAC 184fp). Rabbit antibodies prepared against MAC 182fp immunoprecipitate a Mr 70 000 in vitro translation product from adult mRNA and react in Western blot with a corresponding Mr 70 000 protein present in eggs, cercariae and adult worms but absent in schistosomula. Although the MAC 184fp is not recognized directly by chronic infection or vaccinated mouse antibodies, antisera prepared against the purified fusion protein immunoprecipitate a Mr 27 000 in vitro translation product which also reacts with mouse chronic infection sera. The same Mr 27 000 protein appears to be present in eggs, cercariae, schistosomula and adults as determined by Western blot with rabbit antisera against the MAC 184fp. These results suggest that the S. mansoni polypeptide encoded by the MAC 184 gene, when expressed within a fusion protein, fails to present epitopes normally recognized during natural infection. We propose that these epitopes are conformationally determined and are destroyed when the MAC 184 protein is expressed within β-galactosidase. This abrogation of conformational epitopes may explain the failure of antibodies from chronically infected or vaccinated mice and rabbits to effectively recognize gene products of certain λgt11-fusion protein clones.
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DOI: 10.1016/0166-6851(85)90127-6
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Expression in Escherichia coli of two Schistosoma mansoni genes that encode major antigens recognized by immune mice</title><author><name sortKey="Lanar, David E" sort="Lanar, David E" uniqKey="Lanar D" first="David E." last="Lanar">David E. Lanar</name><affiliation><mods:affiliation>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</mods:affiliation></affiliation></author><author><name sortKey="Pearce, Edward J" sort="Pearce, Edward J" uniqKey="Pearce E" first="Edward J." last="Pearce">Edward J. Pearce</name><affiliation><mods:affiliation>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</mods:affiliation></affiliation></author><author><name sortKey="Sher, Alan" sort="Sher, Alan" uniqKey="Sher A" first="Alan" last="Sher">Alan Sher</name><affiliation><mods:affiliation>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:C009BBE261CDA09B30D9D250ED61DD35C7173A44</idno><date when="1985" year="1985">1985</date><idno type="doi">10.1016/0166-6851(85)90127-6</idno><idno type="url">https://api.istex.fr/document/C009BBE261CDA09B30D9D250ED61DD35C7173A44/fulltext/pdf</idno><idno type="wicri:Area/Istex/Corpus">001928</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a">Expression in Escherichia coli of two Schistosoma mansoni genes that encode major antigens recognized by immune mice</title><author><name sortKey="Lanar, David E" sort="Lanar, David E" uniqKey="Lanar D" first="David E." last="Lanar">David E. Lanar</name><affiliation><mods:affiliation>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</mods:affiliation></affiliation></author><author><name sortKey="Pearce, Edward J" sort="Pearce, Edward J" uniqKey="Pearce E" first="Edward J." last="Pearce">Edward J. Pearce</name><affiliation><mods:affiliation>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</mods:affiliation></affiliation></author><author><name sortKey="Sher, Alan" sort="Sher, Alan" uniqKey="Sher A" first="Alan" last="Sher">Alan Sher</name><affiliation><mods:affiliation>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Molecular & Biochemical Parasitology</title><title level="j" type="abbrev">MOLBIO</title><idno type="ISSN">0166-6851</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1985">1985</date><biblScope unit="volume">17</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="45">45</biblScope><biblScope unit="page" to="60">60</biblScope></imprint><idno type="ISSN">0166-6851</idno></series><idno type="istex">C009BBE261CDA09B30D9D250ED61DD35C7173A44</idno><idno type="DOI">10.1016/0166-6851(85)90127-6</idno><idno type="PII">0166-6851(85)90127-6</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0166-6851</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Two clones which contain genes encoding Schistosoma mansoni proteins recognized by immune mouse sera were chosen from a cDNA λgt11 expression vector library by preselecting clones from the library with rabbit antisera against adult worm phosphate-buffered saline (PBS)-soluble antigens. One clone, MAC 182, codes for part of a Mr 70 000 protein; the other clone, MAC 184, codes for a Mr 27 000 protein. The insert sizes of MAC 182 and MAC 184 are 400 bp and 800 bp, respectively. Both clones express S. mansoni β-galactosidase fusion proteins as products of the construct. Antibodies from either chronically infected mice or mice vaccinated with irradiated cercariae recognize the MAC 182 fusion protein (MAC 182fp) but not the MAC 184 fusion protein (MAC 184fp). Rabbit antibodies prepared against MAC 182fp immunoprecipitate a Mr 70 000 in vitro translation product from adult mRNA and react in Western blot with a corresponding Mr 70 000 protein present in eggs, cercariae and adult worms but absent in schistosomula. Although the MAC 184fp is not recognized directly by chronic infection or vaccinated mouse antibodies, antisera prepared against the purified fusion protein immunoprecipitate a Mr 27 000 in vitro translation product which also reacts with mouse chronic infection sera. The same Mr 27 000 protein appears to be present in eggs, cercariae, schistosomula and adults as determined by Western blot with rabbit antisera against the MAC 184fp. These results suggest that the S. mansoni polypeptide encoded by the MAC 184 gene, when expressed within a fusion protein, fails to present epitopes normally recognized during natural infection. We propose that these epitopes are conformationally determined and are destroyed when the MAC 184 protein is expressed within β-galactosidase. This abrogation of conformational epitopes may explain the failure of antibodies from chronically infected or vaccinated mice and rabbits to effectively recognize gene products of certain λgt11-fusion protein clones.</div></front></TEI><istex><corpusName>elsevier</corpusName><author><json:item><name>David E. Lanar</name><affiliations><json:string>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</json:string></affiliations></json:item><json:item><name>Edward J. Pearce</name><affiliations><json:string>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</json:string></affiliations></json:item><json:item><name>Alan Sher</name><affiliations><json:string>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Schistosoma mansoni</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Mouse immunity</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Expression in Escherichia coli</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Expression vector</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>λgt11 bacteriophage</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>β-Galactosidase-fusion protein</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>cDNA library</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>mRNA</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>DNA</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Antigenic conformational determinants</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>MAC : mansoni adult cDNA</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>MAC 182fp : fusion protein made by MAC 182 λ-clone</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>MAC 184fp : fusion protein made by MAC 184 λ-clone</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>pMAC 182 : pUC 8 plasmid containing schistosome gene insert from MAC 182</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>pMAC 184 : pUC 8 plasmid containing schistosome gene insert from MAC 184</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>NPGB : p-nitro-p′-guanidine benzoate</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>TCLK : N-p′-tosyl l-lysine chloromethylketone</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>TPLK : l-1-tosylamide-2-phenylethyl-chloromethylketone</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>PMSF : phenylmethyl sulphonylfluoride</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>IPTG : isopropylthio-β-d-galactoside</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>SDS-PAGE : sodium dodecyl sulfate polyacrylamide gel electrophoresis</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>TBST : Tris-buffered saline/Tween-20 wash buffer</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>FCS : fetal calf serum</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>SWAP : soluble worm antigen preparation</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>SCHLAP : schistosomula antigen preparation</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>SEA : soluble egg antigen</value></json:item></subject><language><json:string>eng</json:string></language><abstract>Two clones which contain genes encoding Schistosoma mansoni proteins recognized by immune mouse sera were chosen from a cDNA λgt11 expression vector library by preselecting clones from the library with rabbit antisera against adult worm phosphate-buffered saline (PBS)-soluble antigens. One clone, MAC 182, codes for part of a Mr 70 000 protein; the other clone, MAC 184, codes for a Mr 27 000 protein. The insert sizes of MAC 182 and MAC 184 are 400 bp and 800 bp, respectively. Both clones express S. mansoni β-galactosidase fusion proteins as products of the construct. Antibodies from either chronically infected mice or mice vaccinated with irradiated cercariae recognize the MAC 182 fusion protein (MAC 182fp) but not the MAC 184 fusion protein (MAC 184fp). Rabbit antibodies prepared against MAC 182fp immunoprecipitate a Mr 70 000 in vitro translation product from adult mRNA and react in Western blot with a corresponding Mr 70 000 protein present in eggs, cercariae and adult worms but absent in schistosomula. Although the MAC 184fp is not recognized directly by chronic infection or vaccinated mouse antibodies, antisera prepared against the purified fusion protein immunoprecipitate a Mr 27 000 in vitro translation product which also reacts with mouse chronic infection sera. The same Mr 27 000 protein appears to be present in eggs, cercariae, schistosomula and adults as determined by Western blot with rabbit antisera against the MAC 184fp. These results suggest that the S. mansoni polypeptide encoded by the MAC 184 gene, when expressed within a fusion protein, fails to present epitopes normally recognized during natural infection. We propose that these epitopes are conformationally determined and are destroyed when the MAC 184 protein is expressed within β-galactosidase. This abrogation of conformational epitopes may explain the failure of antibodies from chronically infected or vaccinated mice and rabbits to effectively recognize gene products of certain λgt11-fusion protein clones.</abstract><qualityIndicators><score>8.5</score><pdfVersion>1.4</pdfVersion><pdfPageSize>440 x 677 pts</pdfPageSize><refBibsNative>true</refBibsNative><keywordCount>26</keywordCount><abstractCharCount>2012</abstractCharCount><pdfWordCount>10248</pdfWordCount><pdfCharCount>37212</pdfCharCount><pdfPageCount>16</pdfPageCount><abstractWordCount>308</abstractWordCount></qualityIndicators><title>Expression in Escherichia coli of two Schistosoma mansoni genes that encode major antigens recognized by immune mice</title><pii><json:string>0166-6851(85)90127-6</json:string></pii><genre><json:string>research-article</json:string></genre><serie><pages><last>953</last><first>948</first></pages><genre></genre><language><json:string>unknown</json:string></language><title>V. Variation in macrophage schistosomulacidal and tumoricidal activities among mouse strains and correlation with resistance to reinfection</title></serie><host><volume>17</volume><pii><json:string>S0166-6851(00)X0264-2</json:string></pii><pages><last>60</last><first>45</first></pages><issn><json:string>0166-6851</json:string></issn><issue>1</issue><genre><json:string>Journal</json:string></genre><language><json:string>unknown</json:string></language><title>Molecular & Biochemical Parasitology</title><publicationDate>1985</publicationDate></host><categories><wos><json:string>BIOCHEMISTRY & MOLECULAR BIOLOGY</json:string><json:string>PARASITOLOGY</json:string><json:string>BIOLOGY</json:string></wos></categories><publicationDate>1985</publicationDate><copyrightDate>1985</copyrightDate><doi><json:string>10.1016/0166-6851(85)90127-6</json:string></doi><id>C009BBE261CDA09B30D9D250ED61DD35C7173A44</id><fulltext><json:item><original>true</original><mimetype>application/pdf</mimetype><extension>pdf</extension><uri>https://api.istex.fr/document/C009BBE261CDA09B30D9D250ED61DD35C7173A44/fulltext/pdf</uri></json:item><json:item><original>true</original><mimetype>text/plain</mimetype><extension>txt</extension><uri>https://api.istex.fr/document/C009BBE261CDA09B30D9D250ED61DD35C7173A44/fulltext/txt</uri></json:item><json:item><original>false</original><mimetype>application/zip</mimetype><extension>zip</extension><uri>https://api.istex.fr/document/C009BBE261CDA09B30D9D250ED61DD35C7173A44/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/C009BBE261CDA09B30D9D250ED61DD35C7173A44/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a">Expression in Escherichia coli of two Schistosoma mansoni genes that encode major antigens recognized by immune mice</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>ELSEVIER</publisher><availability><p>ELSEVIER</p></availability><date>1985</date></publicationStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">Expression in Escherichia coli of two Schistosoma mansoni genes that encode major antigens recognized by immune mice</title><author><persName><forename type="first">David E.</forename><surname>Lanar</surname></persName><affiliation>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</affiliation></author><author><persName><forename type="first">Edward J.</forename><surname>Pearce</surname></persName><affiliation>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</affiliation></author><author><persName><forename type="first">Alan</forename><surname>Sher</surname></persName><affiliation>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</affiliation></author></analytic><monogr><title level="j">Molecular & Biochemical Parasitology</title><title level="j" type="abbrev">MOLBIO</title><idno type="pISSN">0166-6851</idno><idno type="PII">S0166-6851(00)X0264-2</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1985"></date><biblScope unit="volume">17</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="45">45</biblScope><biblScope unit="page" to="60">60</biblScope></imprint></monogr><idno type="istex">C009BBE261CDA09B30D9D250ED61DD35C7173A44</idno><idno type="DOI">10.1016/0166-6851(85)90127-6</idno><idno type="PII">0166-6851(85)90127-6</idno></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1985</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Two clones which contain genes encoding Schistosoma mansoni proteins recognized by immune mouse sera were chosen from a cDNA λgt11 expression vector library by preselecting clones from the library with rabbit antisera against adult worm phosphate-buffered saline (PBS)-soluble antigens. One clone, MAC 182, codes for part of a Mr 70 000 protein; the other clone, MAC 184, codes for a Mr 27 000 protein. The insert sizes of MAC 182 and MAC 184 are 400 bp and 800 bp, respectively. Both clones express S. mansoni β-galactosidase fusion proteins as products of the construct. Antibodies from either chronically infected mice or mice vaccinated with irradiated cercariae recognize the MAC 182 fusion protein (MAC 182fp) but not the MAC 184 fusion protein (MAC 184fp). Rabbit antibodies prepared against MAC 182fp immunoprecipitate a Mr 70 000 in vitro translation product from adult mRNA and react in Western blot with a corresponding Mr 70 000 protein present in eggs, cercariae and adult worms but absent in schistosomula. Although the MAC 184fp is not recognized directly by chronic infection or vaccinated mouse antibodies, antisera prepared against the purified fusion protein immunoprecipitate a Mr 27 000 in vitro translation product which also reacts with mouse chronic infection sera. The same Mr 27 000 protein appears to be present in eggs, cercariae, schistosomula and adults as determined by Western blot with rabbit antisera against the MAC 184fp. These results suggest that the S. mansoni polypeptide encoded by the MAC 184 gene, when expressed within a fusion protein, fails to present epitopes normally recognized during natural infection. We propose that these epitopes are conformationally determined and are destroyed when the MAC 184 protein is expressed within β-galactosidase. This abrogation of conformational epitopes may explain the failure of antibodies from chronically infected or vaccinated mice and rabbits to effectively recognize gene products of certain λgt11-fusion protein clones.</p></abstract><textClass><keywords scheme="keyword"><list><head>Keywords</head><item><term>Schistosoma mansoni</term></item><item><term>Mouse immunity</term></item><item><term>Expression in Escherichia coli</term></item><item><term>Expression vector</term></item><item><term>λgt11 bacteriophage</term></item><item><term>β-Galactosidase-fusion protein</term></item><item><term>cDNA library</term></item><item><term>mRNA</term></item><item><term>DNA</term></item><item><term>Antigenic conformational determinants</term></item></list></keywords></textClass><textClass><keywords scheme="keyword"><list><head>Abbreviations</head><item><term>MAC</term><term>mansoni adult cDNA</term></item><item><term>MAC 182fp</term><term>fusion protein made by MAC 182 λ-clone</term></item><item><term>MAC 184fp</term><term>fusion protein made by MAC 184 λ-clone</term></item><item><term>pMAC 182</term><term>pUC 8 plasmid containing schistosome gene insert from MAC 182</term></item><item><term>pMAC 184</term><term>pUC 8 plasmid containing schistosome gene insert from MAC 184</term></item><item><term>NPGB</term><term>p-nitro-p′-guanidine benzoate</term></item><item><term>TCLK</term><term>N-p′-tosyl l-lysine chloromethylketone</term></item><item><term>TPLK</term><term>l-1-tosylamide-2-phenylethyl-chloromethylketone</term></item><item><term>PMSF</term><term>phenylmethyl sulphonylfluoride</term></item><item><term>IPTG</term><term>isopropylthio-β-d-galactoside</term></item><item><term>SDS-PAGE</term><term>sodium dodecyl sulfate polyacrylamide gel electrophoresis</term></item><item><term>TBST</term><term>Tris-buffered saline/Tween-20 wash buffer</term></item><item><term>FCS</term><term>fetal calf serum</term></item><item><term>SWAP</term><term>soluble worm antigen preparation</term></item><item><term>SCHLAP</term><term>schistosomula antigen preparation</term></item><item><term>SEA</term><term>soluble egg antigen</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1985-05-31">Registration</change><change when="1985">Published</change></revisionDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla"><item-info><jid>MOLBIO</jid><aid>85901276</aid><ce:pii>0166-6851(85)90127-6</ce:pii><ce:doi>10.1016/0166-6851(85)90127-6</ce:doi><ce:copyright type="unknown" year="1985"></ce:copyright></item-info><head><ce:title>Expression in <ce:italic>Escherichia coli</ce:italic> of two <ce:italic>Schistosoma mansoni</ce:italic> genes that encode major antigens recognized by immune mice</ce:title><ce:author-group><ce:author><ce:given-name>David E.</ce:given-name><ce:surname>Lanar</ce:surname></ce:author><ce:author><ce:given-name>Edward J.</ce:given-name><ce:surname>Pearce</ce:surname></ce:author><ce:author><ce:given-name>Alan</ce:given-name><ce:surname>Sher</ce:surname></ce:author><ce:affiliation><ce:textfn>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</ce:textfn></ce:affiliation></ce:author-group><ce:date-received day="25" month="3" year="1985"></ce:date-received><ce:date-accepted day="31" month="5" year="1985"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Two clones which contain genes encoding <ce:italic>Schistosoma mansoni</ce:italic> proteins recognized by immune mouse sera were chosen from a cDNA λgt11 expression vector library by preselecting clones from the library with rabbit antisera against adult worm phosphate-buffered saline (PBS)-soluble antigens. One clone, MAC 182, codes for part of a <ce:italic>M</ce:italic><ce:inf>r</ce:inf> 70 000 protein; the other clone, MAC 184, codes for a <ce:italic>M</ce:italic><ce:inf>r</ce:inf> 27 000 protein. The insert sizes of MAC 182 and MAC 184 are 400 bp and 800 bp, respectively. Both clones express <ce:italic>S. mansoni</ce:italic> β-galactosidase fusion proteins as products of the construct. Antibodies from either chronically infected mice or mice vaccinated with irradiated cercariae recognize the MAC 182 fusion protein (MAC 182fp) but not the MAC 184 fusion protein (MAC 184fp). Rabbit antibodies prepared against MAC 182fp immunoprecipitate a <ce:italic>M</ce:italic><ce:inf>r</ce:inf> 70 000 in vitro translation product from adult mRNA and react in Western blot with a corresponding <ce:italic>M</ce:italic><ce:inf>r</ce:inf> 70 000 protein present in eggs, cercariae and adult worms but absent in schistosomula. Although the MAC 184fp is not recognized directly by chronic infection or vaccinated mouse antibodies, antisera prepared against the purified fusion protein immunoprecipitate a <ce:italic>M</ce:italic><ce:inf>r</ce:inf> 27 000 in vitro translation product which also reacts with mouse chronic infection sera. The same <ce:italic>M</ce:italic><ce:inf>r</ce:inf> 27 000 protein appears to be present in eggs, cercariae, schistosomula and adults as determined by Western blot with rabbit antisera against the MAC 184fp. These results suggest that the <ce:italic>S. mansoni</ce:italic> polypeptide encoded by the MAC 184 gene, when expressed within a fusion protein, fails to present epitopes normally recognized during natural infection. We propose that these epitopes are conformationally determined and are destroyed when the MAC 184 protein is expressed within β-galactosidase. This abrogation of conformational epitopes may explain the failure of antibodies from chronically infected or vaccinated mice and rabbits to effectively recognize gene products of certain λgt11-fusion protein clones.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text><ce:italic>Schistosoma mansoni</ce:italic></ce:text></ce:keyword><ce:keyword><ce:text>Mouse immunity</ce:text></ce:keyword><ce:keyword><ce:text>Expression in <ce:italic>Escherichia coli</ce:italic></ce:text></ce:keyword><ce:keyword><ce:text>Expression vector</ce:text></ce:keyword><ce:keyword><ce:text>λgt11 bacteriophage</ce:text></ce:keyword><ce:keyword><ce:text>β-Galactosidase-fusion protein</ce:text></ce:keyword><ce:keyword><ce:text>cDNA library</ce:text></ce:keyword><ce:keyword><ce:text>mRNA</ce:text></ce:keyword><ce:keyword><ce:text>DNA</ce:text></ce:keyword><ce:keyword><ce:text>Antigenic conformational determinants</ce:text></ce:keyword></ce:keywords><ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title><ce:keyword><ce:text>MAC</ce:text><ce:keyword><ce:text>mansoni adult cDNA</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>MAC 182fp</ce:text><ce:keyword><ce:text>fusion protein made by MAC 182 λ-clone</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>MAC 184fp</ce:text><ce:keyword><ce:text>fusion protein made by MAC 184 λ-clone</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>pMAC 182</ce:text><ce:keyword><ce:text>pUC 8 plasmid containing schistosome gene insert from MAC 182</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>pMAC 184</ce:text><ce:keyword><ce:text>pUC 8 plasmid containing schistosome gene insert from MAC 184</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>NPGB</ce:text><ce:keyword><ce:text><ce:italic>p</ce:italic>-nitro-<ce:italic>p</ce:italic>′-guanidine benzoate</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>TCLK</ce:text><ce:keyword><ce:text><ce:italic>N</ce:italic>-<ce:italic>p</ce:italic>′-tosyl <ce:small-caps>l</ce:small-caps>-lysine chloromethylketone</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>TPLK</ce:text><ce:keyword><ce:text><ce:small-caps>l</ce:small-caps>-1-tosylamide-2-phenylethyl-chloromethylketone</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>PMSF</ce:text><ce:keyword><ce:text>phenylmethyl sulphonylfluoride</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>IPTG</ce:text><ce:keyword><ce:text>isopropylthio-β-<ce:small-caps>d</ce:small-caps>-galactoside</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>SDS-PAGE</ce:text><ce:keyword><ce:text>sodium dodecyl sulfate polyacrylamide gel electrophoresis</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>TBST</ce:text><ce:keyword><ce:text>Tris-buffered saline/Tween-20 wash buffer</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>FCS</ce:text><ce:keyword><ce:text>fetal calf serum</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>SWAP</ce:text><ce:keyword><ce:text>soluble worm antigen preparation</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>SCHLAP</ce:text><ce:keyword><ce:text>schistosomula antigen preparation</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>SEA</ce:text><ce:keyword><ce:text>soluble egg antigen</ce:text></ce:keyword></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Expression in Escherichia coli of two Schistosoma mansoni genes that encode major antigens recognized by immune mice</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>Expression in</title></titleInfo><name type="personal"><namePart type="given">David E.</namePart><namePart type="family">Lanar</namePart><affiliation>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Edward J.</namePart><namePart type="family">Pearce</namePart><affiliation>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Alan</namePart><namePart type="family">Sher</namePart><affiliation>Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article"></genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1985</dateIssued><dateValid encoding="w3cdtf">1985-05-31</dateValid><copyrightDate encoding="w3cdtf">1985</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType></physicalDescription><abstract lang="en">Two clones which contain genes encoding Schistosoma mansoni proteins recognized by immune mouse sera were chosen from a cDNA λgt11 expression vector library by preselecting clones from the library with rabbit antisera against adult worm phosphate-buffered saline (PBS)-soluble antigens. One clone, MAC 182, codes for part of a Mr 70 000 protein; the other clone, MAC 184, codes for a Mr 27 000 protein. The insert sizes of MAC 182 and MAC 184 are 400 bp and 800 bp, respectively. Both clones express S. mansoni β-galactosidase fusion proteins as products of the construct. Antibodies from either chronically infected mice or mice vaccinated with irradiated cercariae recognize the MAC 182 fusion protein (MAC 182fp) but not the MAC 184 fusion protein (MAC 184fp). Rabbit antibodies prepared against MAC 182fp immunoprecipitate a Mr 70 000 in vitro translation product from adult mRNA and react in Western blot with a corresponding Mr 70 000 protein present in eggs, cercariae and adult worms but absent in schistosomula. Although the MAC 184fp is not recognized directly by chronic infection or vaccinated mouse antibodies, antisera prepared against the purified fusion protein immunoprecipitate a Mr 27 000 in vitro translation product which also reacts with mouse chronic infection sera. The same Mr 27 000 protein appears to be present in eggs, cercariae, schistosomula and adults as determined by Western blot with rabbit antisera against the MAC 184fp. These results suggest that the S. mansoni polypeptide encoded by the MAC 184 gene, when expressed within a fusion protein, fails to present epitopes normally recognized during natural infection. We propose that these epitopes are conformationally determined and are destroyed when the MAC 184 protein is expressed within β-galactosidase. This abrogation of conformational epitopes may explain the failure of antibodies from chronically infected or vaccinated mice and rabbits to effectively recognize gene products of certain λgt11-fusion protein clones.</abstract><subject><genre>Keywords</genre><topic>Schistosoma mansoni</topic><topic>Mouse immunity</topic><topic>Expression in Escherichia coli</topic><topic>Expression vector</topic><topic>λgt11 bacteriophage</topic><topic>β-Galactosidase-fusion protein</topic><topic>cDNA library</topic><topic>mRNA</topic><topic>DNA</topic><topic>Antigenic conformational determinants</topic></subject><subject><genre>Abbreviations</genre><topic>MAC : mansoni adult cDNA</topic><topic>MAC 182fp : fusion protein made by MAC 182 λ-clone</topic><topic>MAC 184fp : fusion protein made by MAC 184 λ-clone</topic><topic>pMAC 182 : pUC 8 plasmid containing schistosome gene insert from MAC 182</topic><topic>pMAC 184 : pUC 8 plasmid containing schistosome gene insert from MAC 184</topic><topic>NPGB : p-nitro-p′-guanidine benzoate</topic><topic>TCLK : N-p′-tosyl l-lysine chloromethylketone</topic><topic>TPLK : l-1-tosylamide-2-phenylethyl-chloromethylketone</topic><topic>PMSF : phenylmethyl sulphonylfluoride</topic><topic>IPTG : isopropylthio-β-d-galactoside</topic><topic>SDS-PAGE : sodium dodecyl sulfate polyacrylamide gel electrophoresis</topic><topic>TBST : Tris-buffered saline/Tween-20 wash buffer</topic><topic>FCS : fetal calf serum</topic><topic>SWAP : soluble worm antigen preparation</topic><topic>SCHLAP : schistosomula antigen preparation</topic><topic>SEA : soluble egg antigen</topic></subject><relatedItem type="host"><titleInfo><title>Molecular & Biochemical Parasitology</title></titleInfo><titleInfo type="abbreviated"><title>MOLBIO</title></titleInfo><genre type="Journal">journal</genre><originInfo><dateIssued encoding="w3cdtf">198510</dateIssued></originInfo><identifier type="ISSN">0166-6851</identifier><identifier type="PII">S0166-6851(00)X0264-2</identifier><part><date>198510</date><detail type="volume"><number>17</number><caption>vol.</caption></detail><detail type="issue"><number>1</number><caption>no.</caption></detail><extent unit="issue pages"><start>1</start><end>130</end></extent><extent unit="pages"><start>45</start><end>60</end></extent></part></relatedItem><identifier type="istex">C009BBE261CDA09B30D9D250ED61DD35C7173A44</identifier><identifier type="DOI">10.1016/0166-6851(85)90127-6</identifier><identifier type="PII">0166-6851(85)90127-6</identifier><recordInfo><recordContentSource>ELSEVIER</recordContentSource></recordInfo></mods></metadata><enrichments><istex:catWosTEI uri="https://api.istex.fr/document/C009BBE261CDA09B30D9D250ED61DD35C7173A44/enrichments/catWos"><teiHeader><profileDesc><textClass><classCode scheme="WOS">BIOCHEMISTRY & MOLECULAR BIOLOGY</classCode><classCode scheme="WOS">PARASITOLOGY</classCode><classCode scheme="WOS">BIOLOGY</classCode></textClass></profileDesc></teiHeader></istex:catWosTEI></enrichments></istex></record>Pour manipuler ce document sous Unix (Dilib)
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