Production of polyclonal antiserum against recombinant VP28 protein and its application for the detection of white spot syndrome virus in crustaceans

Abstract

The VP28 gene of white spot syndrome virus (WSSV) was cloned into pRSET B expression vector. The VP28 protein was expressed as a protein with a 6-histidine taq in Escherichia coli GJ1158 with NaCl induction. Antiserum was raised against this recombinant-VP28 protein in rabbits and it recognized VP28 protein in naturally and experimentally WSSV-infected shrimp, marine crabs, freshwater prawns and freshwater crabs. The antiserum did not recognize any of the other known WSSV structural proteins. Various organs such as eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages were found to be good materials for detection of WSSV using the antiserum and detection of WSSV was successful in experimentally infected Penaeus monodon and P. indicus at 12 and 24 h post-infection (p.i.), respectively. The antiserum was capable of detecting WSSV in 5 ng of total haemolymph protein from WSSV-infected shrimp.

Passive protection of shrimp against white spot syndrome virus (WSSV) using specific antibody from egg yolk of chickens immunized with inactivated virus or a WSSV-DNA vaccine

“…Though invertebrates lack a true adaptive immune response system and seem to rely on various innate immune responses [12], novel research strategies including vaccination against the virus and its structural proteins have been performed. Some previous studies have shown that antiserum against VP28 abolished WSSV-induced mortality of Penaeus monodon [10] and cytopathic effects in P. monodon lymphoid organ cells in vitro [13]. In addition, antibodies against VP28 and fusion proteins comprising portions of VP28 and VP19 have successfully neutralized WSSV [14,15].…”

Tolerance to white spot syndrome virus (WSSV) in the freshwater prawn Macrobrachium rosenbergii is associated with low VP28 envelope protein expression

“…Western blot detection of VP28 in infected samples was conducted as described by Talbot et al (1984). After separation on SDS-PAGE gel, proteins were transferred onto nitrocellulose membranes (Macherey-Nagel) that were subsequently treated overnight at 4°C with blocking buffer followed by incubation with primary antiserum rabbit anti-r-VP28 IgG (diluted to a ratio of 1:2500 in 1% nonfat dry milk/phosphate-buffered saline) that was obtained in our previous study (Yoganandhan et al 2004) for 3 h. Subsequently, the membrane was incubated in ALPconjugated goat anti-rabbit IgG (Sigma) for 1 h, and VP28 was detected using a substrate solution of 4-nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Sigma).…”

Identification of icp11, the most highly expressed gene of shrimp white spot syndrome virus (WSSV)

“…In AmEPV-infected cells, spheroidin alone accounts for up to 30-40% of the total protein of infected insect cells (Winter et al 1995). WSSV also has several highly expressed structural genes/proteins, and, to date, the major structural protein VP28 has been the most commonly used WSSV target for immuno-based detection (Liu et al 2002, Yoganandhan et al 2004. Here, however, we have shown that the transcription expression levels of ICP11 are approximately 3 × greater than those of VP28.…”

“…The sensitivity of this kind of test depends on the immuno-detected target protein, and several candidate proteins have already been conside-Identification of icp11, the most highly expressed gene of shrimp white spot syndrome virus (WSSV) red. Liu et al (2002) and Yoganandhan et al (2004) independently developed procedures that targeted the WSSV major envelope protein, VP28, while Okumura et al (2005) developed a reverse passive latex agglutination method to detect WSSV virion particles by using anti-WSSV immunoglobulin G (IgG). All 3 of these studies were based on WSSV structural proteins, which until now were thought to be the most highly expressed proteins of WSSV.…”