Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique

Peng Yeh Lai, Chia Tse Hsu, Shao Hung Wang, Jin Ching Lee, Min Jen Tseng, Jaulang Hwang, Wen Tsai Ji, Hau Ren Chen

研究成果: 雜誌貢獻文章

2 引文 (Scopus)

摘要

Dengue virus (DENV; genus Flavivirus) contains a positive-stranded RNA genome. Binding of DENV to host cells is mediated through domain III of the viral envelope protein. Many therapeutic mAbs against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named the linear array epitope (LAE) technique for producing monoclone-like polyclonal antibodies. To prove this method could be utilized to produce antibody against epitopes with low antigenicity, a region of 10 aa (V365NIEAEPPFG374) from domain III of the envelope protein in DENV serotype 2 (DENV2) was selected to design the primers for the LAE technique. A DNA fragment encoding 10 directed repeats of these 10 aa for producing the tandem-repeated peptides was obtained and fused with glutathione S-transferase (GST)-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from Escherichia coli and used as antigen for immunizing rabbits to obtain the polyclonal antibody. Furthermore, the EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using Western blot and immunofluorescence assays. Most importantly, this antibody was also able to detect DENV2 virions by ELISA, and could block viral entry into BHK-21 cells as shown by immunofluorescence and quantitative real-time PCR assays. Taken together, the LAE technique could be applied successfully for the production of antibodies against antigens with low antigenicity, and shows high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic applications in the future.

原文英語
頁(從 - 到)2155-2165
頁數11
期刊Journal of General Virology
95
DOIs
出版狀態已發佈 - 十月 1 2014

指紋

Dengue Virus
Neutralizing Antibodies
Epitopes
Antibodies
Proteins
Glutathione Transferase
Fluorescent Antibody Technique
Viral Envelope Proteins
Antigens
Flavivirus
Protein S
Virion
Antibody Formation
Real-Time Polymerase Chain Reaction
Western Blotting
Enzyme-Linked Immunosorbent Assay
Genome
RNA
Escherichia coli
Rabbits

ASJC Scopus subject areas

  • Virology
  • Medicine(all)

引用此文

Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique. / Lai, Peng Yeh; Hsu, Chia Tse; Wang, Shao Hung; Lee, Jin Ching; Tseng, Min Jen; Hwang, Jaulang; Ji, Wen Tsai; Chen, Hau Ren.

於: Journal of General Virology, 卷 95, 01.10.2014, p. 2155-2165.

研究成果: 雜誌貢獻文章

Lai, Peng Yeh ; Hsu, Chia Tse ; Wang, Shao Hung ; Lee, Jin Ching ; Tseng, Min Jen ; Hwang, Jaulang ; Ji, Wen Tsai ; Chen, Hau Ren. / Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique. 於: Journal of General Virology. 2014 ; 卷 95. 頁 2155-2165.
@article{ca6dd0b1031f4b14b87fb842cfb9281c,
title = "Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique",
abstract = "Dengue virus (DENV; genus Flavivirus) contains a positive-stranded RNA genome. Binding of DENV to host cells is mediated through domain III of the viral envelope protein. Many therapeutic mAbs against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named the linear array epitope (LAE) technique for producing monoclone-like polyclonal antibodies. To prove this method could be utilized to produce antibody against epitopes with low antigenicity, a region of 10 aa (V365NIEAEPPFG374) from domain III of the envelope protein in DENV serotype 2 (DENV2) was selected to design the primers for the LAE technique. A DNA fragment encoding 10 directed repeats of these 10 aa for producing the tandem-repeated peptides was obtained and fused with glutathione S-transferase (GST)-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from Escherichia coli and used as antigen for immunizing rabbits to obtain the polyclonal antibody. Furthermore, the EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using Western blot and immunofluorescence assays. Most importantly, this antibody was also able to detect DENV2 virions by ELISA, and could block viral entry into BHK-21 cells as shown by immunofluorescence and quantitative real-time PCR assays. Taken together, the LAE technique could be applied successfully for the production of antibodies against antigens with low antigenicity, and shows high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic applications in the future.",
author = "Lai, {Peng Yeh} and Hsu, {Chia Tse} and Wang, {Shao Hung} and Lee, {Jin Ching} and Tseng, {Min Jen} and Jaulang Hwang and Ji, {Wen Tsai} and Chen, {Hau Ren}",
year = "2014",
month = "10",
day = "1",
doi = "10.1099/vir.0.062562-0",
language = "English",
volume = "95",
pages = "2155--2165",
journal = "Journal of General Virology",
issn = "0022-1317",
publisher = "Society for General Microbiology",

}

TY - JOUR

T1 - Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique

AU - Lai, Peng Yeh

AU - Hsu, Chia Tse

AU - Wang, Shao Hung

AU - Lee, Jin Ching

AU - Tseng, Min Jen

AU - Hwang, Jaulang

AU - Ji, Wen Tsai

AU - Chen, Hau Ren

PY - 2014/10/1

Y1 - 2014/10/1

N2 - Dengue virus (DENV; genus Flavivirus) contains a positive-stranded RNA genome. Binding of DENV to host cells is mediated through domain III of the viral envelope protein. Many therapeutic mAbs against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named the linear array epitope (LAE) technique for producing monoclone-like polyclonal antibodies. To prove this method could be utilized to produce antibody against epitopes with low antigenicity, a region of 10 aa (V365NIEAEPPFG374) from domain III of the envelope protein in DENV serotype 2 (DENV2) was selected to design the primers for the LAE technique. A DNA fragment encoding 10 directed repeats of these 10 aa for producing the tandem-repeated peptides was obtained and fused with glutathione S-transferase (GST)-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from Escherichia coli and used as antigen for immunizing rabbits to obtain the polyclonal antibody. Furthermore, the EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using Western blot and immunofluorescence assays. Most importantly, this antibody was also able to detect DENV2 virions by ELISA, and could block viral entry into BHK-21 cells as shown by immunofluorescence and quantitative real-time PCR assays. Taken together, the LAE technique could be applied successfully for the production of antibodies against antigens with low antigenicity, and shows high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic applications in the future.

AB - Dengue virus (DENV; genus Flavivirus) contains a positive-stranded RNA genome. Binding of DENV to host cells is mediated through domain III of the viral envelope protein. Many therapeutic mAbs against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named the linear array epitope (LAE) technique for producing monoclone-like polyclonal antibodies. To prove this method could be utilized to produce antibody against epitopes with low antigenicity, a region of 10 aa (V365NIEAEPPFG374) from domain III of the envelope protein in DENV serotype 2 (DENV2) was selected to design the primers for the LAE technique. A DNA fragment encoding 10 directed repeats of these 10 aa for producing the tandem-repeated peptides was obtained and fused with glutathione S-transferase (GST)-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from Escherichia coli and used as antigen for immunizing rabbits to obtain the polyclonal antibody. Furthermore, the EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using Western blot and immunofluorescence assays. Most importantly, this antibody was also able to detect DENV2 virions by ELISA, and could block viral entry into BHK-21 cells as shown by immunofluorescence and quantitative real-time PCR assays. Taken together, the LAE technique could be applied successfully for the production of antibodies against antigens with low antigenicity, and shows high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic applications in the future.

UR - http://www.scopus.com/inward/record.url?scp=84907199832&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84907199832&partnerID=8YFLogxK

U2 - 10.1099/vir.0.062562-0

DO - 10.1099/vir.0.062562-0

M3 - Article

C2 - 24948392

AN - SCOPUS:84907199832

VL - 95

SP - 2155

EP - 2165

JO - Journal of General Virology

JF - Journal of General Virology

SN - 0022-1317

ER -