Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector
Production of structural proteins from foot-and-mouth disease virus (FMDV) and bovine herpes virus (BHV-1) in Nicotiana benthamiana through the use of a tobacco mosaic virus-based vector (TMV-30B) has been reported previously. The development of the TMV-30B-HISc vector, a new version that adds a C-terminal histidine (His) sequence to the foreign protein expressed is described. Coding sequences from the FMDV VPl protein and the core protein, p24, from a clade C HIV-1 isolate from Zambia were cloned into the new vector and infective RNAs were generated for each construct to inoculate N. benthamiana plants. His-tagged proteins were purified from inoculated leaves using immobilized metal affinity chromatography (IMAC) as detected by Coomassie blue staining and proteins were further characterized in Western blot assays using a commercial anti-6xHis mAb and specific polyclonal antisera for each protein. While yields obtained for the VPl-His protein after purification were similar to those in crude extracts obtained with the previous TMV-VPl vector, p24-His yields were 10-15 times higher than those of VPl-His. Twenty-five grams of TMV-p24-HISc inoculated leaves were processed to obtain 2.5 mg of isolated p24-His and the recombinant protein was inoculated in rabbits to test immunogenicity and antigenic integrity of the plant-produced p24-His. Animals developed a strong and specific humoral response to the p24-His after the first booster and immune sera was able to recognize the native p24 from a different clade expressed on the surface of the HIV-1 chronically infected HUT78/ARV T-cell line. Importantly, the recombinant p24-His proved its efficiency by confirming the serology of 117 samples previously tested by two rapid HIV-1 tests, thus representing an excellent alternative for production of highly specific diagnostic reagents for HIV endemic regions in the developing world. © 2004 Elsevier B.V. All rights reserved.
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2004
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Subjects: | HIV, Plant expression, Diagnosis, TMV-vector, Immune response, |
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dig-inia-es-10261-2943372023-02-20T10:37:43Z Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector Pérez-Filgueira, D. M. Brayfield, B. P. Phiri, S. Borca, M. V. Wood, C. Morris, T. J. HIV Plant expression Diagnosis TMV-vector Immune response Production of structural proteins from foot-and-mouth disease virus (FMDV) and bovine herpes virus (BHV-1) in Nicotiana benthamiana through the use of a tobacco mosaic virus-based vector (TMV-30B) has been reported previously. The development of the TMV-30B-HISc vector, a new version that adds a C-terminal histidine (His) sequence to the foreign protein expressed is described. Coding sequences from the FMDV VPl protein and the core protein, p24, from a clade C HIV-1 isolate from Zambia were cloned into the new vector and infective RNAs were generated for each construct to inoculate N. benthamiana plants. His-tagged proteins were purified from inoculated leaves using immobilized metal affinity chromatography (IMAC) as detected by Coomassie blue staining and proteins were further characterized in Western blot assays using a commercial anti-6xHis mAb and specific polyclonal antisera for each protein. While yields obtained for the VPl-His protein after purification were similar to those in crude extracts obtained with the previous TMV-VPl vector, p24-His yields were 10-15 times higher than those of VPl-His. Twenty-five grams of TMV-p24-HISc inoculated leaves were processed to obtain 2.5 mg of isolated p24-His and the recombinant protein was inoculated in rabbits to test immunogenicity and antigenic integrity of the plant-produced p24-His. Animals developed a strong and specific humoral response to the p24-His after the first booster and immune sera was able to recognize the native p24 from a different clade expressed on the surface of the HIV-1 chronically infected HUT78/ARV T-cell line. Importantly, the recombinant p24-His proved its efficiency by confirming the serology of 117 samples previously tested by two rapid HIV-1 tests, thus representing an excellent alternative for production of highly specific diagnostic reagents for HIV endemic regions in the developing world. © 2004 Elsevier B.V. All rights reserved. 2023-02-20T10:37:43Z 2023-02-20T10:37:43Z 2004 journal article Journal of Virological Methods 121(2): 201-208 (2004) 0166-0934 http://hdl.handle.net/20.500.12792/3448 http://hdl.handle.net/10261/294337 10.1016/j.jviromet.2004.06.022 en none Elsevier |
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HIV Plant expression Diagnosis TMV-vector Immune response HIV Plant expression Diagnosis TMV-vector Immune response Pérez-Filgueira, D. M. Brayfield, B. P. Phiri, S. Borca, M. V. Wood, C. Morris, T. J. Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector |
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Production of structural proteins from foot-and-mouth disease virus (FMDV) and bovine herpes virus (BHV-1) in Nicotiana benthamiana through the use of a tobacco mosaic virus-based vector (TMV-30B) has been reported previously. The development of the TMV-30B-HISc vector, a new version that adds a C-terminal histidine (His) sequence to the foreign protein expressed is described. Coding sequences from the FMDV VPl protein and the core protein, p24, from a clade C HIV-1 isolate from Zambia were cloned into the new vector and infective RNAs were generated for each construct to inoculate N. benthamiana plants. His-tagged proteins were purified from inoculated leaves using immobilized metal affinity chromatography (IMAC) as detected by Coomassie blue staining and proteins were further characterized in Western blot assays using a commercial anti-6xHis mAb and specific polyclonal antisera for each protein. While yields obtained for the VPl-His protein after purification were similar to those in crude extracts obtained with the previous TMV-VPl vector, p24-His yields were 10-15 times higher than those of VPl-His. Twenty-five grams of TMV-p24-HISc inoculated leaves were processed to obtain 2.5 mg of isolated p24-His and the recombinant protein was inoculated in rabbits to test immunogenicity and antigenic integrity of the plant-produced p24-His. Animals developed a strong and specific humoral response to the p24-His after the first booster and immune sera was able to recognize the native p24 from a different clade expressed on the surface of the HIV-1 chronically infected HUT78/ARV T-cell line. Importantly, the recombinant p24-His proved its efficiency by confirming the serology of 117 samples previously tested by two rapid HIV-1 tests, thus representing an excellent alternative for production of highly specific diagnostic reagents for HIV endemic regions in the developing world. © 2004 Elsevier B.V. All rights reserved. |
format |
journal article |
topic_facet |
HIV Plant expression Diagnosis TMV-vector Immune response |
author |
Pérez-Filgueira, D. M. Brayfield, B. P. Phiri, S. Borca, M. V. Wood, C. Morris, T. J. |
author_facet |
Pérez-Filgueira, D. M. Brayfield, B. P. Phiri, S. Borca, M. V. Wood, C. Morris, T. J. |
author_sort |
Pérez-Filgueira, D. M. |
title |
Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector |
title_short |
Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector |
title_full |
Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector |
title_fullStr |
Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector |
title_full_unstemmed |
Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector |
title_sort |
preserved antigenicity of hiv-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (tmv)-derived vector |
publisher |
Elsevier |
publishDate |
2004 |
url |
http://hdl.handle.net/20.500.12792/3448 http://hdl.handle.net/10261/294337 |
work_keys_str_mv |
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