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Potato virus Y recombinant coat protein production in Escherichia coli : Applications in antibody production and virus detection
Author(s) -
Rabie Mona,
Abdel Aleem Engy E.,
Elwan Esraa A.,
Fattouh Faiza A.
Publication year - 2021
Publication title -
journal of phytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.53
H-Index - 60
eISSN - 1439-0434
pISSN - 0931-1785
DOI - 10.1111/jph.12997
Subject(s) - polyclonal antibodies , biology , recombinant dna , antiserum , microbiology and biotechnology , escherichia coli , blot , expression vector , antibody , affinity chromatography , lac operon , gel electrophoresis , virology , virus , biochemistry , enzyme , gene , immunology
The coat protein coding region of Potato virus Y (PVY) was cloned into pGEM‐T, sub‐cloned into plasmid pET‐28a (+) then transformed into Escherichia coli strain BL21 (DE3) pLysS to express the protein. Induction of the recombinant protein was made with isopropyl‐β‐ d ‐thiogalactopyranoside (IPTG), which has been tested in a variety of concentrations and for varying periods of time. The highest expression level of the protein was achieved with 1 mM IPTG for 4 hr. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) followed by Western blotting has been used to confirm the identity of the expressed protein. The PVY coat protein (30 kDa) was purified under denaturing conditions by affinity chromatography and identity reconfirmed by Western blotting. The expressed protein was used as a recombinant antigen to raise polyclonal antibodies in mice. Purified anti‐PVYCP immunoglobulin (IgG) reacted positively with PVY‐infected leaf samples and the purified expressed CP in plate‐trapped antigen enzyme‐linked immunosorbent assay (PTA‐ELISA). The currently produced antiserum detects the more prevailing strains in the region: PVY N, NTN and Wilga.