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. 2023 Mar;13(3):358-364.
doi: 10.5455/OVJ.2023.v13.i3.13. Epub 2023 Mar 19.

Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies

Rosario Liberti  1 , Claudia Colabella  1 , Lucia Anzalone  1 , Giulio Severi  1 , Antonella Di Paolo  1 , Cristina Casciari  1 , Anna Beatrice Casano  2 , Monica Giammarioli  1 , Monica Cagiola  1 , Francesco Feliziani  1 , Antonio De Giuseppe  1
Affiliations

Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies

Rosario Liberti et al. Open Vet J. 2023 Mar.

Abstract

Background: African Swine Fever (ASF) is an infectious disease that affects domestic pig and wild boar populations. The ASF Virus (ASFV) has a genome characterized by a very complex DNA (170-193 kb) that encodes for more than 200 different proteins. Among these, the highly immunogenic phosphoprotein p30 plays a fundamental role in the induction of specific antibodies. To date, the lack of a vaccine against the disease requires continuous studies to improve knowledge about the virus and the development of new tests in addition to virological ones.

Aim: The aim of this work was to produce specific monoclonal antibodies (mAbs) against the p30 protein of ASFV, which could find useful applications in routine diagnostics and the implementation of new diagnostic tools.

Methods: ASFV p30 encoding gene was amplified and used for the generation of the recombinant baculovirus by transfection of the Sf21 insect cells. The recombinant protein was analyzed by immunofluorescence assay, purified, and used for mice Balb-c immunization. The hybridomas obtained were cultured and screened, using an indirect Enzyme-linked Immunosorbent Assay (iELISA), in order to select clones that secrete the mAbs of interest.

Results: The expression of recombinant p30 protein was assessed using direct immunofluorescence. The purified p30 protein fractions were analyzed by Coomassie gels staining confirming the presence of bands with a molecular weight of 30 kDa and used for the immunization of Balb-c mice. Six clones of pure hybridomas secreting the specific mAbs against recombinant p30 were obtained and tested in iELISA. The mAbs were also characterized by Western blot and immunofluorescence assay. The best results were obtained with the anti-p30 mAb 2B8E10 clone which showed high reactivity with both recombinant and viral p30 protein, respectively.

Conclusion: In this work, a recombinant p30 protein produced in an insect cell system was purified and used to immunize Balb-c mice. Six anti-p30 mAbs-secreting hybridomas clone cells were obtained. These mAbs displayed high reactivity against the recombinant protein, but only 2B8E10 mAb showed excellent functionality against the p30 protein produced by ASFV. These results open the possibility to develop different diagnostic assays.

Keywords: ASFV; ASFV p30 protein; Baculovirus; Monoclonal antibody.

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Conflict of interest statement

The Authors declare that there is no conflict of interest.

Figures

Fig. 1.
Fig. 1.. Immunofluorescence assay of p30 recombinant protein expressed in Sf21 cells using anti-p30 FITC mAb: (A) infected Sf21 cells; (B) uninfected Sf21 cells (negative control).
Fig. 2.
Fig. 2.. SDS-PAGE and Coomassie gel staining of purified recombinant p30 protein; recombinant protein elution fractions (lanes 1–7); protein standard molecular weight is reported.
Fig. 3.
Fig. 3.. Western Blot analysis of anti-p30 mAbs using purified recombinant p30. (A) mAb 2B8E10; (B) mAb 2B8D8B1; (C) mAb 2B8G5D2; (D) mAb 7C3E3G5; (E) mAb 4G6F4; (F) mAb 4F11C2. Recombinant p30 protein (lane 1) and uninfected Sf21 cells (lane 2). Protein standard molecular weight is reported (spectra multicolor low range protein ladder).
Fig. 4.
Fig. 4.. Western Blot analysis of anti-p30 mAbs. (A) mAb 2B8E10; (B) mAb 2B8D8B1; (C) mAb 2B8G5D2; (D) mAb 7C3E3G5; (E) mAb 4G6F4; (F) mAb 4F11C2. ASFV infected vero cells (lane 1) and uninfected Vero cells (lane 2). Protein standard molecular weight is reported (spectra multicolor low range protein ladder).
Fig. 5.
Fig. 5.. Immunofluorescence assay carried out using anti p30 mAbs on ASFV infected Vero cells. (A) mAb 2B8E10; (B) mAb 2B8D8B1; (C) mAb 2B8G5D3; (D) mAb 7C3E3G5; (E) mAb 4G6F4; (F) mAb 4F11C2; (G) uninfected Vero cells.
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