SrasV1, Main, Exploration, bibRecord, 004527

[Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity].

Identifieur interne : 004527 ( Main/Exploration ); précédent : 004526; suivant : 004528

[Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity].

Auteurs : Ru-Shi Liu [République populaire de Chine] ; Yi-Lan Qiu ; Kun-Yu Yang ; Zhi-Hong Zhang ; Liang Liang ; Jun Zhang ; Ning-Shao Xia

Source :

Sheng wu gong cheng xue bao = Chinese journal of biotechnology [ 1000-3061 ] ; 2005.

RBID : pubmed:16176089

Descripteurs français

KwdFr :
- Clonage moléculaire, Pichia (génétique), Pichia (métabolisme), Protéines nucléocapside (biosynthèse), Protéines nucléocapside (génétique), Protéines nucléocapside (immunologie), Protéines recombinantes (biosynthèse), Protéines recombinantes (génétique), Protéines recombinantes (immunologie), Virus du SRAS (génétique).
MESH :
- biosynthèse : Protéines nucléocapside, Protéines recombinantes.
- génétique : Pichia, Protéines nucléocapside, Protéines recombinantes, Virus du SRAS.
- immunologie : Protéines nucléocapside, Protéines recombinantes.
- métabolisme : Pichia.
- Clonage moléculaire.

English descriptors

KwdEn :
- Cloning, Molecular, Nucleocapsid Proteins (biosynthesis), Nucleocapsid Proteins (genetics), Nucleocapsid Proteins (immunology), Pichia (genetics), Pichia (metabolism), Recombinant Proteins (biosynthesis), Recombinant Proteins (genetics), Recombinant Proteins (immunology), SARS Virus (genetics).
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

Affiliations:

République populaire de Chine

Le document en format XML

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<author><name sortKey="Zhang, Jun" sort="Zhang, Jun" uniqKey="Zhang J" first="Jun" last="Zhang">Jun Zhang</name>
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<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>
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<term>Protéines nucléocapside (génétique)</term>
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<term>Protéines recombinantes (génétique)</term>
<term>Protéines recombinantes (immunologie)</term>
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<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>
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[Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity].

[Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity].

Source :

Descripteurs français

English descriptors

Abstract

Links toward previous steps (curation, corpus...)

Le document en format XML

Pour manipuler ce document sous Unix (Dilib)

Pour mettre un lien sur cette page dans le réseau Wicri

Pour générer des pages wiki

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