Development and Application of the Binding Assay for Tomato Spotted Wilt Virus Nucleoprotein Using Fluorescent Polarization Technology

doi:10.13523/j.cb.20181103

China Biotechnology

2018, Vol. 38

Issue (11): 18-24 DOI: 10.13523/j.cb.20181103

Development and Application of the Binding Assay for Tomato Spotted Wilt Virus Nucleoprotein Using Fluorescent Polarization Technology

Jing-li WANG¹,Zhen-zhen DING³,Hui LIU¹,Yan-ting TANG^2,^***(

)

^1. College of Pharmacy, Nankai University, Tianjin 300071, China
^2. Tianjin International Joint Academy of Biomedicine, Tianjin 300457, China
^3. Dynamiker Biotechnology (Tianjin) Co. Ltd., Tianjin 300467, China

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Abstract

Objective:Based on fluorescence polarization technology, a binding assay targeting the interaction between tomato spotted wilt virus (TSWV) nucleoprotein (NP) and nucleic acid was established and used for drug screening.Methods:The full-length NP DNA amplicon was cloned into the pGEX-6p-1 expression vector, and the construct pGEX-6p-1-NP was transformed into E. coli strain BL(DE3). Recombinant NP protein was purified with a general protocol. A fluorescence polarization assay that is sensitive to inhibitors disrupting TSWV NP/nucleic acid interactions was established. The assay’s DMSO tolerance, incubation time, stability and variability were studied and a pilot drug screening was performed.Results: The recombinant plasmid pGEX-6p-1-NP was successfully constructed and the high quality recombinant NP was purified. A 384-well fluorescence polarization assay targeting TSWV NP and nucleic acid interaction was developed and validated, with a signal-to-noise ratio of 8:1 and a Z factor of 0.82 was obtained, demonstrating the assay is HTS compatible. The assay was used to screen 1 000 compounds in the chemical libraries. After the primary screening, one compound with IC₅₀ of 4.146μmol/L was identified. Conclusion:The fluorescence polarization assay is stable and suitable for the screening inhibitors blocking the interaction between NP and nucleic acids, and the compound provides a reference for the prevention and control of TSWV.

Key words： Tomato spotted wilt virus Nucleoprotein Fluorescence polarization Drug screening

Received: 05 March 2018 Published: 06 December 2018

ZTFLH:

Q819

Corresponding Authors: Yan-ting TANG E-mail: titihere@163.com

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Cite this article:

Jing-li WANG,Zhen-zhen DING,Hui LIU,Yan-ting TANG. Development and Application of the Binding Assay for Tomato Spotted Wilt Virus Nucleoprotein Using Fluorescent Polarization Technology. China Biotechnology, 2018, 38(11): 18-24.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20181103 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I11/18

Fig.1 FITC labeled 25T

Fig.2 The recombinant NP was analyzed by chromatogram and 12% SDS-PAGE(a) Ion exchange chromatogram (b) Size exclusion chromatogram (c) 12% SDS-PAGE

Fig.3 The relativity analysis between probe concentration and fluorescence intensity (a) The linear regression analysis between parallel polarized light intensity and probe concentration (R²=0.992 1) (b) Polarization values at various probe concentrations

Table 1 The average value of fluorescence intensity and polarization values at various probe concentrations

Table 2 K_d values at different incubation times

Fig.4 Saturation binding curve of the labeled 25T to NP (a) The polarization value increases with increasing protein concentration (b) The polarization value changes with the increase of protein concentration (logarithmic value)

Table 3 IC₅₀ values at different incubation times

Fig.5 Competitive binding curve by titration with the unlabeled probe 25T at different incubation times, keeping the labeled probe and NP concentration constant

Table 4 Polarization values and K_d values at vatious DMSO concentrations

Fig.6 DMSO tolerance of the assay (a) Effect of DMSO on the polarization values (b) Effect of DMSO on the K_d

Table 5 The components of the FP Assay

Fig.7 Calculation of Z factor to monitor assay performance, by polarization values of the negative control group and the positive control group


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