Eukaryotic expression of NS1 major antigen region of PPV and development of an indirect ELISA based on the expressed protein

To construct secretory expression vector of PPV NS1 gene, the fragment of PPV NS1 gene coding for major antigen region of the NS1 protein was amplified by PCR and inserted into multiple clone site of eukaryotic expression vector pPICZalpha-A. The recombinant pPICZalpha-A-NS1 plasmid was transferred...

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Bibliographic Details
Published inBingdu xuebao Vol. 28; no. 6; p. 628
Main Authors Ma, Hui, Zhao, Xu-Yong, Bian, Chuan-Zhou
Format Journal Article
LanguageChinese
Published China 01.11.2012
Subjects
Animals
Antibodies, Viral - immunology
Antigens, Viral - genetics
Antigens, Viral - immunology
Enzyme-Linked Immunosorbent Assay - methods
Escherichia coli - genetics
Escherichia coli - metabolism
Gene Expression
Parvoviridae Infections - diagnosis
Parvoviridae Infections - immunology
Parvoviridae Infections - veterinary
Parvoviridae Infections - virology
Parvovirus, Porcine - genetics
Parvovirus, Porcine - immunology
Parvovirus, Porcine - isolation & purification
Recombinant Proteins - genetics
Recombinant Proteins - immunology
Swine
Swine Diseases - diagnosis
Swine Diseases - immunology
Swine Diseases - virology
Viral Nonstructural Proteins - genetics
Viral Nonstructural Proteins - immunology
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Summary:To construct secretory expression vector of PPV NS1 gene, the fragment of PPV NS1 gene coding for major antigen region of the NS1 protein was amplified by PCR and inserted into multiple clone site of eukaryotic expression vector pPICZalpha-A. The recombinant pPICZalpha-A-NS1 plasmid was transferred into P. pastoris strain GS115 mediated by electro transform. Recombinant P. pastoris strain GS115 was induced to express the fusion protein by methanol. The expressed and purified protein was analyzed by SDS-PAGE and Western Blot. The recombinant protein was highly-expressed and showed a good immunoreactivity. The indirect ELISA method was developed for detecting antibodies against PPV by checkerboard titration assay. The result showed that the optimal concentration of coated antigen was 3.2 microg/mL and the best dilution of serum was 1 : 80. The positive cut-off value of the ELISA assay was OD450 > 0.4 and OD450 positive serum/OD450 negative serum > 2.0. Compared with HI and commercial ELISA kits, the assay revea
ISSN:1000-8721