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Production of a Recombinant Vp1 Protein of the Chicken Anaemiavirus for the Development of an Enzyme-Linked Immunosorbent Assay

VP1 gene is an important gene responsible for the expression of a protein that may facilitate the diagnosis of chicken anaemia virus (CAV) infection.The VP1 gene was cloned and expressed in prokaryotic expression vector pRSET–B.The VP1 protein expression in E. coli was achieved as a fusion protein...

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Main Author: Mahgoub, Elhem Omer
Format: Thesis
Language:English
English
Published: 2004
Online Access:http://psasir.upm.edu.my/id/eprint/189/1/570123_t_fpv_2005_14.pdf
http://psasir.upm.edu.my/id/eprint/189/
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spelling my.upm.eprints.1892013-05-27T06:46:22Z http://psasir.upm.edu.my/id/eprint/189/ Production of a Recombinant Vp1 Protein of the Chicken Anaemiavirus for the Development of an Enzyme-Linked Immunosorbent Assay Mahgoub, Elhem Omer VP1 gene is an important gene responsible for the expression of a protein that may facilitate the diagnosis of chicken anaemia virus (CAV) infection.The VP1 gene was cloned and expressed in prokaryotic expression vector pRSET–B.The VP1 protein expression in E. coli was achieved as a fusion protein containing an immunogenic epitope. The protein expressed was detected by anti-VP1 monoclonal antibody with an apparent molecular weight of 50 kDa. Batch fermentation was used to scale up production of the protein in E. coli.The recombinant VP1 protein was successfully expressed during high cell density culture. The use of tangential flow filtration (TFF) filtration step for dialysis and desalting increased both the specific activity and the final yield of the purified fraction.The protein expressed has been tested as an antigen for detection of antibody to CAV in infected chicken.An enzyme-linked immunosorbent assay (ELISA) applied for the detection of serum antibody to CAV.This test depends on the availability of CAV polyclonal antibodies present in convalescent chicken serum to react with the VP1 antigen adsorbed to the ELISA plate.When serum samples from infected chicken were tested,the sensitivity of the assay was found to be greater than 93.3%.Furthermore, the ELISA test requires serum samples to be diluted at 1:100 compared to 1:50 for commercial ELISA,an increase in the potential of non-specific background that could be contributed by proteins available in serum samples.In conclusion the indirect ELISA approach using VP1 fusion protein has many advantages. The results of the present study can be used as a basis for the development of a reliable ELISA assay for detection of CAV 2004-11 Thesis NonPeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/189/1/570123_t_fpv_2005_14.pdf Mahgoub, Elhem Omer (2004) Production of a Recombinant Vp1 Protein of the Chicken Anaemiavirus for the Development of an Enzyme-Linked Immunosorbent Assay. Masters thesis, Universiti Putra Malaysia. English
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
English
description VP1 gene is an important gene responsible for the expression of a protein that may facilitate the diagnosis of chicken anaemia virus (CAV) infection.The VP1 gene was cloned and expressed in prokaryotic expression vector pRSET–B.The VP1 protein expression in E. coli was achieved as a fusion protein containing an immunogenic epitope. The protein expressed was detected by anti-VP1 monoclonal antibody with an apparent molecular weight of 50 kDa. Batch fermentation was used to scale up production of the protein in E. coli.The recombinant VP1 protein was successfully expressed during high cell density culture. The use of tangential flow filtration (TFF) filtration step for dialysis and desalting increased both the specific activity and the final yield of the purified fraction.The protein expressed has been tested as an antigen for detection of antibody to CAV in infected chicken.An enzyme-linked immunosorbent assay (ELISA) applied for the detection of serum antibody to CAV.This test depends on the availability of CAV polyclonal antibodies present in convalescent chicken serum to react with the VP1 antigen adsorbed to the ELISA plate.When serum samples from infected chicken were tested,the sensitivity of the assay was found to be greater than 93.3%.Furthermore, the ELISA test requires serum samples to be diluted at 1:100 compared to 1:50 for commercial ELISA,an increase in the potential of non-specific background that could be contributed by proteins available in serum samples.In conclusion the indirect ELISA approach using VP1 fusion protein has many advantages. The results of the present study can be used as a basis for the development of a reliable ELISA assay for detection of CAV
format Thesis
author Mahgoub, Elhem Omer
spellingShingle Mahgoub, Elhem Omer
Production of a Recombinant Vp1 Protein of the Chicken Anaemiavirus for the Development of an Enzyme-Linked Immunosorbent Assay
author_facet Mahgoub, Elhem Omer
author_sort Mahgoub, Elhem Omer
title Production of a Recombinant Vp1 Protein of the Chicken Anaemiavirus for the Development of an Enzyme-Linked Immunosorbent Assay
title_short Production of a Recombinant Vp1 Protein of the Chicken Anaemiavirus for the Development of an Enzyme-Linked Immunosorbent Assay
title_full Production of a Recombinant Vp1 Protein of the Chicken Anaemiavirus for the Development of an Enzyme-Linked Immunosorbent Assay
title_fullStr Production of a Recombinant Vp1 Protein of the Chicken Anaemiavirus for the Development of an Enzyme-Linked Immunosorbent Assay
title_full_unstemmed Production of a Recombinant Vp1 Protein of the Chicken Anaemiavirus for the Development of an Enzyme-Linked Immunosorbent Assay
title_sort production of a recombinant vp1 protein of the chicken anaemiavirus for the development of an enzyme-linked immunosorbent assay
publishDate 2004
url http://psasir.upm.edu.my/id/eprint/189/1/570123_t_fpv_2005_14.pdf
http://psasir.upm.edu.my/id/eprint/189/
_version_ 1643821757275045888
score 13.211869

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