DNA versus protein immunisation for production of monoclonal antibodies against Choristoneura fumiferana ecdysone receptor (CfEcR)

Dan Hui Yang; Amina Makhmoudova; Basil M. Arif; Qili Feng; Arthur Retnakaran; Subba Reddy Palli; Peter J. Krell

doi:10.1016/j.vaccine.2006.01.050

DNA versus protein immunisation for production of monoclonal antibodies against Choristoneura fumiferana ecdysone receptor (CfEcR)

Dan Hui Yang, Amina Makhmoudova, Basil M. Arif, Qili Feng, Arthur Retnakaran, Subba Reddy Palli, Peter J. Krell

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

Abstract

The full-length ecdysone receptor cDNA of Choristoneura fumiferana (CfEcR-B) was cloned into bacterial expression systems and the recombinant protein was expressed either with a His-tag (His-EcR-B) or glutathione-S- transferase (GST) fusion (GST-EcR-B). The His-EcR-B was expressed mostly as insoluble aggregates, while the GST-EcR-B was partially soluble and could be purified using affinity chromatography. Mice were then immunised with the purified GST-EcR-B protein. Due to the time-consuming protein expression and purification procedures and the solubility problem of the recombinant protein, we also inserted the full-length CfEcR-B cDNA into the mammalian DNA vaccine expression vector, pVAC1-mcs for DNA immunisation. In vitro expression of CfEcR-B in mammalian cells transfected with the pVAC-EcR-B plasmid was confirmed prior to the delivery of the DNA vaccine into mice. The anti-CfEcR-B MAbs generated from both DNA and protein vaccines were characterised and shown to recognise native CfEcR-B protein induced by 20E in CF-203 insect cells. DNA immunisation was shown to overcome the solubility problem of the bacterial expressed EcR and created a more direct route for monoclonal antibody production for this receptor protein obviating the need for EcR expression and purification to generate the antigen.

Original language	English
Pages (from-to)	3115-3126
Number of pages	12
Journal	Vaccine
Volume	24
Issue number	16
DOIs	https://doi.org/10.1016/j.vaccine.2006.01.050
State	Published - Apr 12 2006

Bibliographical note

Funding Information:
Contributors: We are grateful to Drs. Alan G. Wildeman and Mhairi Skinner (Department of Molecular and Cellular Biology, University of Guelph) for providing mammalian cell lines used in this study. We thank Ms. Annette Morrison (Central Animal Facility, University of Guelph) for technical help on mouse injections and bleeding. We also thank Drs. Frances Sharom and Roselynn Stevenson (Department of Molecular and Cellular Biology, University of Guelph) for use of the FluorChem 8900 (Alpha Innotech) and the Fluorescence Microscope (FM), respectively, in their laboratories, Ms. Dilya Kamalova and Mr. Steve Lord for technical assistance. This work was supported by grants to Dr. Peter Krell from NSERC, Canadian Forestry Service, Rohm and Haas Company, Ontario Genomics Institute and Genome Canada, and Biocontrol Canada.

Keywords

Choristoneura fumiferana
DNA vaccine
Ecdysteroid receptor

ASJC Scopus subject areas

Molecular Medicine
General Immunology and Microbiology
General Veterinary
Public Health, Environmental and Occupational Health
Infectious Diseases

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.vaccine.2006.01.050

Cite this

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title = "DNA versus protein immunisation for production of monoclonal antibodies against Choristoneura fumiferana ecdysone receptor (CfEcR)",

abstract = "The full-length ecdysone receptor cDNA of Choristoneura fumiferana (CfEcR-B) was cloned into bacterial expression systems and the recombinant protein was expressed either with a His-tag (His-EcR-B) or glutathione-S- transferase (GST) fusion (GST-EcR-B). The His-EcR-B was expressed mostly as insoluble aggregates, while the GST-EcR-B was partially soluble and could be purified using affinity chromatography. Mice were then immunised with the purified GST-EcR-B protein. Due to the time-consuming protein expression and purification procedures and the solubility problem of the recombinant protein, we also inserted the full-length CfEcR-B cDNA into the mammalian DNA vaccine expression vector, pVAC1-mcs for DNA immunisation. In vitro expression of CfEcR-B in mammalian cells transfected with the pVAC-EcR-B plasmid was confirmed prior to the delivery of the DNA vaccine into mice. The anti-CfEcR-B MAbs generated from both DNA and protein vaccines were characterised and shown to recognise native CfEcR-B protein induced by 20E in CF-203 insect cells. DNA immunisation was shown to overcome the solubility problem of the bacterial expressed EcR and created a more direct route for monoclonal antibody production for this receptor protein obviating the need for EcR expression and purification to generate the antigen.",

keywords = "Choristoneura fumiferana, DNA vaccine, Ecdysteroid receptor",

author = "Yang, {Dan Hui} and Amina Makhmoudova and Arif, {Basil M.} and Qili Feng and Arthur Retnakaran and Palli, {Subba Reddy} and Krell, {Peter J.}",

note = "Funding Information: Contributors: We are grateful to Drs. Alan G. Wildeman and Mhairi Skinner (Department of Molecular and Cellular Biology, University of Guelph) for providing mammalian cell lines used in this study. We thank Ms. Annette Morrison (Central Animal Facility, University of Guelph) for technical help on mouse injections and bleeding. We also thank Drs. Frances Sharom and Roselynn Stevenson (Department of Molecular and Cellular Biology, University of Guelph) for use of the FluorChem 8900 (Alpha Innotech) and the Fluorescence Microscope (FM), respectively, in their laboratories, Ms. Dilya Kamalova and Mr. Steve Lord for technical assistance. This work was supported by grants to Dr. Peter Krell from NSERC, Canadian Forestry Service, Rohm and Haas Company, Ontario Genomics Institute and Genome Canada, and Biocontrol Canada.",

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doi = "10.1016/j.vaccine.2006.01.050",

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AU - Yang, Dan Hui

AU - Makhmoudova, Amina

AU - Arif, Basil M.

AU - Feng, Qili

AU - Retnakaran, Arthur

AU - Palli, Subba Reddy

AU - Krell, Peter J.

N1 - Funding Information: Contributors: We are grateful to Drs. Alan G. Wildeman and Mhairi Skinner (Department of Molecular and Cellular Biology, University of Guelph) for providing mammalian cell lines used in this study. We thank Ms. Annette Morrison (Central Animal Facility, University of Guelph) for technical help on mouse injections and bleeding. We also thank Drs. Frances Sharom and Roselynn Stevenson (Department of Molecular and Cellular Biology, University of Guelph) for use of the FluorChem 8900 (Alpha Innotech) and the Fluorescence Microscope (FM), respectively, in their laboratories, Ms. Dilya Kamalova and Mr. Steve Lord for technical assistance. This work was supported by grants to Dr. Peter Krell from NSERC, Canadian Forestry Service, Rohm and Haas Company, Ontario Genomics Institute and Genome Canada, and Biocontrol Canada.

PY - 2006/4/12

Y1 - 2006/4/12

N2 - The full-length ecdysone receptor cDNA of Choristoneura fumiferana (CfEcR-B) was cloned into bacterial expression systems and the recombinant protein was expressed either with a His-tag (His-EcR-B) or glutathione-S- transferase (GST) fusion (GST-EcR-B). The His-EcR-B was expressed mostly as insoluble aggregates, while the GST-EcR-B was partially soluble and could be purified using affinity chromatography. Mice were then immunised with the purified GST-EcR-B protein. Due to the time-consuming protein expression and purification procedures and the solubility problem of the recombinant protein, we also inserted the full-length CfEcR-B cDNA into the mammalian DNA vaccine expression vector, pVAC1-mcs for DNA immunisation. In vitro expression of CfEcR-B in mammalian cells transfected with the pVAC-EcR-B plasmid was confirmed prior to the delivery of the DNA vaccine into mice. The anti-CfEcR-B MAbs generated from both DNA and protein vaccines were characterised and shown to recognise native CfEcR-B protein induced by 20E in CF-203 insect cells. DNA immunisation was shown to overcome the solubility problem of the bacterial expressed EcR and created a more direct route for monoclonal antibody production for this receptor protein obviating the need for EcR expression and purification to generate the antigen.

AB - The full-length ecdysone receptor cDNA of Choristoneura fumiferana (CfEcR-B) was cloned into bacterial expression systems and the recombinant protein was expressed either with a His-tag (His-EcR-B) or glutathione-S- transferase (GST) fusion (GST-EcR-B). The His-EcR-B was expressed mostly as insoluble aggregates, while the GST-EcR-B was partially soluble and could be purified using affinity chromatography. Mice were then immunised with the purified GST-EcR-B protein. Due to the time-consuming protein expression and purification procedures and the solubility problem of the recombinant protein, we also inserted the full-length CfEcR-B cDNA into the mammalian DNA vaccine expression vector, pVAC1-mcs for DNA immunisation. In vitro expression of CfEcR-B in mammalian cells transfected with the pVAC-EcR-B plasmid was confirmed prior to the delivery of the DNA vaccine into mice. The anti-CfEcR-B MAbs generated from both DNA and protein vaccines were characterised and shown to recognise native CfEcR-B protein induced by 20E in CF-203 insect cells. DNA immunisation was shown to overcome the solubility problem of the bacterial expressed EcR and created a more direct route for monoclonal antibody production for this receptor protein obviating the need for EcR expression and purification to generate the antigen.

KW - Choristoneura fumiferana

KW - DNA vaccine

KW - Ecdysteroid receptor

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DNA versus protein immunisation for production of monoclonal antibodies against Choristoneura fumiferana ecdysone receptor (CfEcR)

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

UN SDGs

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