Polyclonal antibodies against the recombinantly expressed coat protein of the Citrus psorosis virus
| Autor: | Mohamed S. Salama, Gamal Osman, Reda Salem, Ibrahim A. Arif |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0106 biological sciences
0301 basic medicine Citrus psorosis virus Immunodiagnostics Expression vector biology biology.organism_classification 01 natural sciences Virology law.invention Serology 03 medical and health sciences 030104 developmental biology Antigen lcsh:Biology (General) Polyclonal antibodies law biology.protein Recombinant DNA Antibody General Agricultural and Biological Sciences lcsh:QH301-705.5 010606 plant biology & botany |
| Zdroj: | Saudi Journal of Biological Sciences, Vol 25, Iss 4, Pp 733-738 (2018) |
| Popis: | Psorosis is a damaging disease of citrus that is widespread in many parts of the world. Citrus psorosis virus (CPsV), the type species of the genus Ophiovirus, is the putative causal agent of psorosis. Detection of CPsV by laboratory methods, serology in particular is a primary requirement for large-scale surveys but their production has been impaired by the difficulty of obtaining sufficient clean antigen for immunization. Specific PAbs against coat protein were produced in E. coli using recombinant DNA approach. The full length CP gene fragment was amplified by RT-PCR using total RNA extracted from CPsV infected citrus leaves and CP specific primers. The obtained product (1320bp) was cloned, sequenced and sub-cloned into pET-30(+) expression vector. Expression was induced and screened in different bacterial clones by the presence of the expressed protein (48kDa) and optimized in one clone. Expressed CP was purified using batch chromatography under denaturing conditions. Specificity of expressed protein was demonstrated by ELISA before used as antigen for raising PAbs in mice. Specificity of the raised PAbs to CPsV was verified by ELISA and western blotting. The raised PAbs were showed highly effectiveness in screening by ELISA comparing with the commercial antibodies purchased from Agritest, Valanzano, Italy.The expression of CPsV CP gene in E. coli, production of PAbs using recombinant protein as an antigen, the suitability of these antibodies for use in immunodiagnostics against the CPsV Egyptian isolate have been accomplished in this work. Keywords: CPsV, CP, PAbs, RT-PCR, ELISA, Western blotting |
| Databáze: | OpenAIRE |
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