Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies
Background: African Swine Fever (ASF) is an infectious disease that affects domestic pig and wild boar populations. The African Swine Fever 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 immunogen...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Tripoli University
2023-06-01
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| Series: | Open Veterinary Journal |
| Subjects: | |
| Online Access: | http://www.ejmanager.com/fulltextpdf.php?mno=120515 |
| _version_ | 1797854950661816320 |
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| author | Rosrio Liberti Claudia Colabella Lucia Anzalone Giulio Severi Antonella Di Paolo Cristina Casciari Anna Beatrice Casano Monica Giammarioli Monica Cagiola Francesco Feliziani Antonio De Giuseppe |
| author_facet | Rosrio Liberti Claudia Colabella Lucia Anzalone Giulio Severi Antonella Di Paolo Cristina Casciari Anna Beatrice Casano Monica Giammarioli Monica Cagiola Francesco Feliziani Antonio De Giuseppe |
| author_sort | Rosrio Liberti |
| collection | DOAJ |
| description | Background:
African Swine Fever (ASF) is an infectious disease that affects domestic pig and wild boar populations. The African Swine Fever 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. [Open Vet J 2023; 13(3.000): 358-364] |
| first_indexed | 2024-04-09T20:15:39Z |
| format | Article |
| id | doaj.art-abc57de1c0cb476899be75851aa07ac6 |
| institution | Directory Open Access Journal |
| issn | 2226-4485 |
| language | English |
| last_indexed | 2024-04-09T20:15:39Z |
| publishDate | 2023-06-01 |
| publisher | Tripoli University |
| record_format | Article |
| series | Open Veterinary Journal |
| spelling | doaj.art-abc57de1c0cb476899be75851aa07ac62023-03-31T10:18:09ZengTripoli UniversityOpen Veterinary Journal2226-44852023-06-0113335836410.5455/OVJ.2023.v13.i3.13120515Expression of a recombinant ASFV P30 protein and production of monoclonal antibodiesRosrio Liberti0Claudia Colabella1Lucia Anzalone2Giulio Severi3Antonella Di Paolo4Cristina Casciari5Anna Beatrice Casano6Monica Giammarioli7Monica Cagiola8Francesco Feliziani9Antonio De Giuseppe10Istituto Zooprofilattico Sperimentale dell'Umbria e Marche "Togo Rosati", via G. Salvemini, 1 06126 Perugia Istituto Zooprofilattico Sperimentale dell'Umbria e Marche "Togo Rosati", via G. Salvemini, 1 06126 Perugia Istituto Zooprofilattico Sperimentale dell'Umbria e Marche "Togo Rosati", via G. Salvemini, 1 06126 Perugia Istituto Zooprofilattico Sperimentale dell'Umbria e Marche "Togo Rosati", via G. Salvemini, 1 06126 Perugia Istituto Zooprofilattico Sperimentale dell'Umbria e Marche "Togo Rosati", via G. Salvemini, 1 06126 Perugia Istituto Zooprofilattico Sperimentale dell'Umbria e Marche "Togo Rosati", via G. Salvemini, 1 06126 Perugia Dipartimento di Medicina Veterinaria, Università di Perugia, via San Costanzo 06126 Perugia Istituto Zooprofilattico Sperimentale dell'Umbria e Marche "Togo Rosati", via G. Salvemini, 1 06126 Perugia Istituto Zooprofilattico Sperimentale dell'Umbria e Marche "Togo Rosati", via G. Salvemini, 1 06126 Perugia Istituto Zooprofilattico Sperimentale dell'Umbria e Marche "Togo Rosati", via G. Salvemini, 1 06126 Perugia Istituto Zooprofilattico Sperimentale dell'Umbria e Marche "Togo Rosati", via G. Salvemini, 1 06126 PerugiaBackground: African Swine Fever (ASF) is an infectious disease that affects domestic pig and wild boar populations. The African Swine Fever 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. [Open Vet J 2023; 13(3.000): 358-364]http://www.ejmanager.com/fulltextpdf.php?mno=120515asfvasfv p30 proteinbaculovirusmonoclonal antibody |
| spellingShingle | Rosrio Liberti Claudia Colabella Lucia Anzalone Giulio Severi Antonella Di Paolo Cristina Casciari Anna Beatrice Casano Monica Giammarioli Monica Cagiola Francesco Feliziani Antonio De Giuseppe Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies Open Veterinary Journal asfv asfv p30 protein baculovirus monoclonal antibody |
| title | Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies |
| title_full | Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies |
| title_fullStr | Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies |
| title_full_unstemmed | Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies |
| title_short | Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies |
| title_sort | expression of a recombinant asfv p30 protein and production of monoclonal antibodies |
| topic | asfv asfv p30 protein baculovirus monoclonal antibody |
| url | http://www.ejmanager.com/fulltextpdf.php?mno=120515 |
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