Proteomic Analysis of Cytoskeleton Proteins in Fish

Abstract

In this chapter, we describe laboratory protocols for rearing fish and a simple and efficient method of extracting and identifying pathogen and host proteins that may be involved in entry and replication of commercially important fish viruses. We have used the common carp (Cyprinus carpio L.) and goldfish (Cyprinus auratus) as a model system for studies of proteins involved in viral entry and replication. The chapter describes detailed protocols for maintenance of carp, cell culture, antibody purification of proteins, and use of electrospray-ionization mass spectrometry analysis to screen and identify cytoskeleton and other proteins that may be involved in viral infection and propagation in fish.

Elucidation of putative binding partners for the protein encoded by ORF149 of cyprinid herpesvirus 3 in goldfish (Carassius auratus)

“…Furthermore, the immunogenic and vaccine potentials of several epitopes of CyHV‐3 have been investigated, including Orf12 (Kattlun, Menanteau‐Ledouble, & El‐Matbouli, 2014) which is readily recognized by the immune system of carp and Orf81 for which conflicting evidence exists (Kattlun et al, 2016; Zhou et al, 2014), although little such research has been conducted in goldfish ( Carassius auratus ). In previous reports, our group elucidated pathogen–host interactions in CyHV‐3‐infected C. auratus through the use of monoclonal antibody‐linked pulldown assay followed by electro‐spray ionization mass spectrometry (ESI‐MS) as described in Gotesman, Menanteau‐Ledouble, and El‐Matbouli (2016). C. auratus is a non‐symptomatic carrier of CyHV‐3, and previous studies demonstrated that in C. auratus , several host defence proteins interact with CyHV‐3 (Bergmann et al, 2010; Gotesman, Abd‐Elfattah, Kattlun, Soliman, & El‐Matbouli, 2014).…”

“…Pulldown assays use antibodies to capture a “bait” protein in an affinity resin, and in the present study, we applied a pulldown assay to investigate the proteins interacting with the Orf149 epitope: Orf149 mAbs were linked to N‐hydroxysuccinimide (NHS)‐activated agarose columns (Gotesman et al, 2016) to capture and identify host proteins interacting with CyHV‐3. Kidney samples of C. auratus previously infected by intraperitoneal injection with 200 µl of CyHV‐3 virus at a concentration of 10 4 TCID 50 (Kattlun et al, 2014, 2016) were lysed using a Tissue Lyser (Qiagen).…”

A new age in AquaMedicine: unconventional approach in studying aquatic diseases

“…Differential transcriptional patterns obtained from qPCR based methods [ 99 , 100 ] can be coupled to protein-based studies [ 101 ] to enhance our understanding of pathogen-host interactions. For protein purification, antibodies raised against the pathogen(s) of interest can be used to capture proteins involved in host-pathogen interactions which are subsequently identified by mass spectrometry [ 102 ]. Such studies have been used to differentiate between host pathogen interactions of susceptible carp versus carrier goldfish for CyHV-3 entry and replication [ 103 , 104 ].…”