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Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis

In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of th...

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Autores principales: Huang, Chen-Ji, Peng, Hwei-Ling, Cheng, Chih-Yu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi Publishing Corporation 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3166572/
https://www.ncbi.nlm.nih.gov/pubmed/21904443
http://dx.doi.org/10.1155/2011/359042
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author Huang, Chen-Ji
Peng, Hwei-Ling
Cheng, Chih-Yu
author_facet Huang, Chen-Ji
Peng, Hwei-Ling
Cheng, Chih-Yu
author_sort Huang, Chen-Ji
collection PubMed
description In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W(84), P(95), P(110), or V(129). The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3) were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W(84)S, P(110)S and V(129)L) exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 μM(−1). Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection.
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spelling pubmed-31665722011-09-08 Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis Huang, Chen-Ji Peng, Hwei-Ling Cheng, Chih-Yu J Biomed Biotechnol Research Article In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W(84), P(95), P(110), or V(129). The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3) were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W(84)S, P(110)S and V(129)L) exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 μM(−1). Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection. Hindawi Publishing Corporation 2011 2011-08-29 /pmc/articles/PMC3166572/ /pubmed/21904443 http://dx.doi.org/10.1155/2011/359042 Text en Copyright © 2011 Chen-Ji Huang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Huang, Chen-Ji
Peng, Hwei-Ling
Cheng, Chih-Yu
Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis
title Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis
title_full Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis
title_fullStr Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis
title_full_unstemmed Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis
title_short Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis
title_sort improving antigenicity of the recombinant hepatitis c virus core protein via random mutagenesis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3166572/
https://www.ncbi.nlm.nih.gov/pubmed/21904443
http://dx.doi.org/10.1155/2011/359042
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