[Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity].
Identifieur interne : 002531 ( PubMed/Corpus ); précédent : 002530; suivant : 002532[Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity].
Auteurs : Ru-Shi Liu ; Yi-Lan Qiu ; Kun-Yu Yang ; Zhi-Hong Zhang ; Liang Liang ; Jun Zhang ; Ning-Shao XiaSource :
- Sheng wu gong cheng xue bao = Chinese journal of biotechnology [ 1000-3061 ] ; 2005.
English descriptors
- KwdEn :
- MESH :
- chemical , biosynthesis : Nucleocapsid Proteins, Recombinant Proteins.
- chemical , genetics : Nucleocapsid Proteins, Recombinant Proteins.
- chemical , immunology : Nucleocapsid Proteins, Recombinant Proteins.
- genetics : Pichia, SARS Virus.
- metabolism : Pichia.
- Cloning, Molecular.
Abstract
The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.
PubMed: 16176089
Links to Exploration step
pubmed:16176089Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">[Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity].</title>
<author><name sortKey="Liu, Ru Shi" sort="Liu, Ru Shi" uniqKey="Liu R" first="Ru-Shi" last="Liu">Ru-Shi Liu</name>
<affiliation><nlm:affiliation>Department of Biochemistry and Molecular Biology Life Science College, Hunan Normal University, Changsha 410081, China.</nlm:affiliation>
</affiliation>
</author>
<author><name sortKey="Qiu, Yi Lan" sort="Qiu, Yi Lan" uniqKey="Qiu Y" first="Yi-Lan" last="Qiu">Yi-Lan Qiu</name>
</author>
<author><name sortKey="Yang, Kun Yu" sort="Yang, Kun Yu" uniqKey="Yang K" first="Kun-Yu" last="Yang">Kun-Yu Yang</name>
</author>
<author><name sortKey="Zhang, Zhi Hong" sort="Zhang, Zhi Hong" uniqKey="Zhang Z" first="Zhi-Hong" last="Zhang">Zhi-Hong Zhang</name>
</author>
<author><name sortKey="Liang, Liang" sort="Liang, Liang" uniqKey="Liang L" first="Liang" last="Liang">Liang Liang</name>
</author>
<author><name sortKey="Zhang, Jun" sort="Zhang, Jun" uniqKey="Zhang J" first="Jun" last="Zhang">Jun Zhang</name>
</author>
<author><name sortKey="Xia, Ning Shao" sort="Xia, Ning Shao" uniqKey="Xia N" first="Ning-Shao" last="Xia">Ning-Shao Xia</name>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PubMed</idno>
<date when="2005">2005</date>
<idno type="RBID">pubmed:16176089</idno>
<idno type="pmid">16176089</idno>
<idno type="wicri:Area/PubMed/Corpus">002531</idno>
<idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002531</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en">[Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity].</title>
<author><name sortKey="Liu, Ru Shi" sort="Liu, Ru Shi" uniqKey="Liu R" first="Ru-Shi" last="Liu">Ru-Shi Liu</name>
<affiliation><nlm:affiliation>Department of Biochemistry and Molecular Biology Life Science College, Hunan Normal University, Changsha 410081, China.</nlm:affiliation>
</affiliation>
</author>
<author><name sortKey="Qiu, Yi Lan" sort="Qiu, Yi Lan" uniqKey="Qiu Y" first="Yi-Lan" last="Qiu">Yi-Lan Qiu</name>
</author>
<author><name sortKey="Yang, Kun Yu" sort="Yang, Kun Yu" uniqKey="Yang K" first="Kun-Yu" last="Yang">Kun-Yu Yang</name>
</author>
<author><name sortKey="Zhang, Zhi Hong" sort="Zhang, Zhi Hong" uniqKey="Zhang Z" first="Zhi-Hong" last="Zhang">Zhi-Hong Zhang</name>
</author>
<author><name sortKey="Liang, Liang" sort="Liang, Liang" uniqKey="Liang L" first="Liang" last="Liang">Liang Liang</name>
</author>
<author><name sortKey="Zhang, Jun" sort="Zhang, Jun" uniqKey="Zhang J" first="Jun" last="Zhang">Jun Zhang</name>
</author>
<author><name sortKey="Xia, Ning Shao" sort="Xia, Ning Shao" uniqKey="Xia N" first="Ning-Shao" last="Xia">Ning-Shao Xia</name>
</author>
</analytic>
<series><title level="j">Sheng wu gong cheng xue bao = Chinese journal of biotechnology</title>
<idno type="ISSN">1000-3061</idno>
<imprint><date when="2005" type="published">2005</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Cloning, Molecular</term>
<term>Nucleocapsid Proteins (biosynthesis)</term>
<term>Nucleocapsid Proteins (genetics)</term>
<term>Nucleocapsid Proteins (immunology)</term>
<term>Pichia (genetics)</term>
<term>Pichia (metabolism)</term>
<term>Recombinant Proteins (biosynthesis)</term>
<term>Recombinant Proteins (genetics)</term>
<term>Recombinant Proteins (immunology)</term>
<term>SARS Virus (genetics)</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Nucleocapsid Proteins</term>
<term>Recombinant Proteins</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Nucleocapsid Proteins</term>
<term>Recombinant Proteins</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>Nucleocapsid Proteins</term>
<term>Recombinant Proteins</term>
</keywords>
<keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Pichia</term>
<term>SARS Virus</term>
</keywords>
<keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Pichia</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Cloning, Molecular</term>
</keywords>
</textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en">The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.</div>
</front>
</TEI>
<pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">16176089</PMID>
<DateCompleted><Year>2009</Year>
<Month>07</Month>
<Day>22</Day>
</DateCompleted>
<DateRevised><Year>2005</Year>
<Month>09</Month>
<Day>22</Day>
</DateRevised>
<Article PubModel="Print"><Journal><ISSN IssnType="Print">1000-3061</ISSN>
<JournalIssue CitedMedium="Print"><Volume>21</Volume>
<Issue>4</Issue>
<PubDate><Year>2005</Year>
<Month>Jul</Month>
</PubDate>
</JournalIssue>
<Title>Sheng wu gong cheng xue bao = Chinese journal of biotechnology</Title>
<ISOAbbreviation>Sheng Wu Gong Cheng Xue Bao</ISOAbbreviation>
</Journal>
<ArticleTitle>[Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity].</ArticleTitle>
<Pagination><MedlinePgn>540-6</MedlinePgn>
</Pagination>
<Abstract><AbstractText>The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.</AbstractText>
</Abstract>
<AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Liu</LastName>
<ForeName>Ru-Shi</ForeName>
<Initials>RS</Initials>
<AffiliationInfo><Affiliation>Department of Biochemistry and Molecular Biology Life Science College, Hunan Normal University, Changsha 410081, China.</Affiliation>
</AffiliationInfo>
</Author>
<Author ValidYN="Y"><LastName>Qiu</LastName>
<ForeName>Yi-Lan</ForeName>
<Initials>YL</Initials>
</Author>
<Author ValidYN="Y"><LastName>Yang</LastName>
<ForeName>Kun-Yu</ForeName>
<Initials>KY</Initials>
</Author>
<Author ValidYN="Y"><LastName>Zhang</LastName>
<ForeName>Zhi-Hong</ForeName>
<Initials>ZH</Initials>
</Author>
<Author ValidYN="Y"><LastName>Liang</LastName>
<ForeName>Liang</ForeName>
<Initials>L</Initials>
</Author>
<Author ValidYN="Y"><LastName>Zhang</LastName>
<ForeName>Jun</ForeName>
<Initials>J</Initials>
</Author>
<Author ValidYN="Y"><LastName>Xia</LastName>
<ForeName>Ning-Shao</ForeName>
<Initials>NS</Initials>
</Author>
</AuthorList>
<Language>chi</Language>
<PublicationTypeList><PublicationType UI="D004740">English Abstract</PublicationType>
<PublicationType UI="D016428">Journal Article</PublicationType>
<PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType>
</PublicationTypeList>
</Article>
<MedlineJournalInfo><Country>China</Country>
<MedlineTA>Sheng Wu Gong Cheng Xue Bao</MedlineTA>
<NlmUniqueID>9426463</NlmUniqueID>
<ISSNLinking>1000-3061</ISSNLinking>
</MedlineJournalInfo>
<ChemicalList><Chemical><RegistryNumber>0</RegistryNumber>
<NameOfSubstance UI="D019590">Nucleocapsid Proteins</NameOfSubstance>
</Chemical>
<Chemical><RegistryNumber>0</RegistryNumber>
<NameOfSubstance UI="D011994">Recombinant Proteins</NameOfSubstance>
</Chemical>
<Chemical><RegistryNumber>0</RegistryNumber>
<NameOfSubstance UI="C099602">nucleocapsid protein, Coronavirus</NameOfSubstance>
</Chemical>
</ChemicalList>
<CitationSubset>IM</CitationSubset>
<MeshHeadingList><MeshHeading><DescriptorName UI="D003001" MajorTopicYN="N">Cloning, Molecular</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D019590" MajorTopicYN="N">Nucleocapsid Proteins</DescriptorName>
<QualifierName UI="Q000096" MajorTopicYN="Y">biosynthesis</QualifierName>
<QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName>
<QualifierName UI="Q000276" MajorTopicYN="N">immunology</QualifierName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D010843" MajorTopicYN="N">Pichia</DescriptorName>
<QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName>
<QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D011994" MajorTopicYN="N">Recombinant Proteins</DescriptorName>
<QualifierName UI="Q000096" MajorTopicYN="N">biosynthesis</QualifierName>
<QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName>
<QualifierName UI="Q000276" MajorTopicYN="Y">immunology</QualifierName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D045473" MajorTopicYN="N">SARS Virus</DescriptorName>
<QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName>
</MeshHeading>
</MeshHeadingList>
</MedlineCitation>
<PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2005</Year>
<Month>9</Month>
<Day>24</Day>
<Hour>9</Hour>
<Minute>0</Minute>
</PubMedPubDate>
<PubMedPubDate PubStatus="medline"><Year>2009</Year>
<Month>7</Month>
<Day>23</Day>
<Hour>9</Hour>
<Minute>0</Minute>
</PubMedPubDate>
<PubMedPubDate PubStatus="entrez"><Year>2005</Year>
<Month>9</Month>
<Day>24</Day>
<Hour>9</Hour>
<Minute>0</Minute>
</PubMedPubDate>
</History>
<PublicationStatus>ppublish</PublicationStatus>
<ArticleIdList><ArticleId IdType="pubmed">16176089</ArticleId>
</ArticleIdList>
</PubmedData>
</pubmed>
</record>
Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/SrasV1/Data/PubMed/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 002531 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/PubMed/Corpus/biblio.hfd -nk 002531 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien |wiki= Sante |area= SrasV1 |flux= PubMed |étape= Corpus |type= RBID |clé= pubmed:16176089 |texte= [Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity]. }}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/PubMed/Corpus/RBID.i -Sk "pubmed:16176089" \ | HfdSelect -Kh $EXPLOR_AREA/Data/PubMed/Corpus/biblio.hfd \ | NlmPubMed2Wicri -a SrasV1
![]() | This area was generated with Dilib version V0.6.33. | ![]() |