Production of a polyclonal antibody to the VP26 nucleocapsid protein of white spot syndrome virus (wssv) and its use as a biosensor

doi:10.1007/s11705-012-1289-y

Front Chem Sci Eng

2012, Vol. 6

Issue (2) : 216-223 https://doi.org/10.1007/s11705-012-1289-y

RESEARCH ARTICLE

Production of a polyclonal antibody to the VP26 nucleocapsid protein of white spot syndrome virus (wssv) and its use as a biosensor

Suchera LOYPRASERT-THANANIMIT^1,2(

), Akrapon SALEEDANG², Proespichaya KANATHARANA^3,4, Panote THAVARUNGKUL^3,5, Wilaiwan CHOTIGEAT^1,2

1. Center for Genomics and Bioinformatics Research, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand; 2. Department of Molecular Biotechnology and Bioinformatics, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand; 3. Trace Analysis and Biosensor Research Center, Prince of Songkla University, Songkhla 90112, Thailand; 4. Department of Chemistry, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand; 5. Department of Physics, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand

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Abstract

White spot syndrome virus (WSSV) is a major cause of high mortality in cultured shrimp all over the world. VP26 is one of the structural proteins of WSSV that is assumed to assist in recognizing its host and assists the viral nucleocapsid to move toward the nucleus of the host cell. The objective of this work was to produce a polyclonal antibody against VP26 and use it as a biosensor. The recombinant VP26 protein (rVP26) was produced in E. coli (BL21), purified and used for immunizing rabbits to obtain a polyclonal antibody. Western blot analysis confirmed that the antiserum had a specific immunoreactivity to the VP26 of WSSV. This VP26 antiserum was immobilized onto a gold electrode for use as the sensing surface to detect WSSV under a flow injection system. The impedance change in the presence of VP26 was monitored in real time. The sensitivity of the biosensor was in the linear range of 160–160000 copies of WSSV, indicating that it is good and sensitive for analysis of WSSV. The specificity of the biosensor was supported by the observation that no impedance change was detected even at high concentrations when using Yellow Head Virus (YHV). This biosensor may be applied to monitor the amount of WSSV in water during shrimp cultivation.

Keywords recombinant protein polyclonal antibody label-free biosensor impedance white spot syndrome virus (WSSV)

Corresponding Author(s): LOYPRASERT-THANANIMIT Suchera,Email:suchera.l@psu.ac.th, loyprasert@yahoo.com

Issue Date: 05 June 2012

Cite this article:

Suchera LOYPRASERT-THANANIMIT,Akrapon SALEEDANG,Proespichaya KANATHARANA, et al. Production of a polyclonal antibody to the VP26 nucleocapsid protein of white spot syndrome virus (wssv) and its use as a biosensor[J]. Front Chem Sci Eng, 2012, 6(2): 216-223.

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https://academic.hep.com.cn/fcse/EN/10.1007/s11705-012-1289-y
https://academic.hep.com.cn/fcse/EN/Y2012/V6/I2/216

Fig.1 Schematic diagram showing the label-free impedimetric biosensor (a) and the impedance response when the antisera VP26 was immobilized on the modified electrode. The base-line signal of the carrier buffer is recorded initially. After injection of WSSV, the association of the immunocomplex between the VP26 antigen on the WSSV and antibody causes the impedance (″) to increase (b)

Fig.2 (a) Coomassie brilliant blue stained 14% SDS-PAGE gel and (b) Western blot using anti-VP26 polyclonal antibody for detecting GST and GST-VP26 protein expressed in (HB2151). Lane M: protein marker; lane 1: lysate of bacteria containing pGEX plasmid before induction with 1 mmol?L IPTG; lane 2: lysate of bacteria containing pGEX plasmid after induction with 1 mmol?L IPTG; lane 3: lysate of bacteria containing pGEX-VP26 plasmid before induction with 1 mmol?L IPTG; lane 4: lysate of bacteria containing pGEX-VP26 plasmid after induction with 1 mmol?L IPTG; lane 5: GST purified protein (29 kDa); lane 6: GST-VP26 purified protein (55 kDa)

Fig.3 Impedance change caused by the binding of different dilutions of WSSV stock solution to immobilized antisera VP26 on the gold electrode

Fig.4 Impedance signal with the other samples to test their binding to the immobilized antibody on the gold electrode

Fig.5 Reproducibility of the response obtained from the label-free biosensor by continuous injection of 1600 copies of WSSV after regeneration (HCl, pH 2.5) between each individual assay

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