Development of polyclonal antiserum against movement protein from Potato leafroll virus and its application for the virus detection

Abstract

The serological method is one of the most important techniques extensively used in crop production to detect different pathogens, especially plant viruses. An antiserum is essential for serological tests. The 17 kDa movement protein (MP) of Potato leafroll virus (PLRV) is related to the membranous structures and localized to the plasmodesmata, but there is no report on preparation of PLRV-MP antiserum for detection of PLRV. To prepare PLRV-MP antiserum, reverse transcription polymerase chain reaction (RT-PCR) was carried out to amplify the PLRV-MP gene, which was constructed into a prokaryotic vector to express the protein in Escherichia coli for immunization of rabbits, after purification. Western blotting revealed that this developed antiserum could effectively detect PLRV, but with better results from the perspective of color development and economics by using antiserum at the ratio range of 1:10000 to 1:40000, presenting high sensitivity and specificity to PLRV. The serological detection results for PLRV of the field samples were identical to the RT-PCR detection. This is the first report on the development of PLRV-MP antiserum that has been successfully used for both laboratory and field detection of PLRV. The results provide a fundamental tool for further research on the function of PLRV-MP.

The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection

“…Western blot analysis was performed using the rabbit anti-flag antibody (Sigma-Aldrich, St Louis, MO, USA) diluted at 1:1000, or mouse anti-flag antibody (Sigma-Aldrich) diluted at 1:5000, or rabbit anti-GFP antibody (GenScript, Nanjing, China) diluted at 1:3000, or rabbit anti-PLRV movement protein (MP) antibody [ 45 ] diluted at 1:5000, as a primary antibody. Incubation was then performed with goat anti-rabbit IgG (Sigma-Aldrich) diluted at 1:3000, or goat anti-mouse HRP (Bio-Rad) diluted at 1:3000, as a secondary antibody.…”

Development of polyclonal antisera against movement proteins of the three poleroviruses infecting cucurbits and their serological relationships

“…In regards to the serological detection, only antiserum against CABYV prepared by purified virion was once used in Western blotting or ELISA [4]. Some recent studies showed that polyclonal antiserum against MP can detect the accumulation of virus from the family Luteoviridae [40][41][42][43]. However, there are no reports on either preparing polyclonal antiserum against MP of the three viruses or the serological relationships among them.…”