Proteomic analysis of differentially expressed proteins in Fenneropenaeus chinensis hemocytes upon white spot syndrome virus infection

Abstract

To elucidate molecular responses of shrimp hemocytes to white spot syndrome virus (WSSV) infection, two-dimensional gel electrophoresis was applied to investigate differentially expressed proteins in hemocytes of Chinese shrimp (Fenneropenaeus chinensis) at 24 h post infection (hpi). Approximately 580 protein spots were detected in hemocytes of healthy and WSSV-infected shrimps. Quantitative intensity analysis revealed 26 protein spots were significantly up-regulated, and 19 spots were significantly down-regulated. By mass spectrometry, small ubiquitin-like modifier (SUMO) 1, cytosolic MnSOD, triosephosphate isomerase, tubulin alpha-1 chain, microtubule-actin cross-linking factor 1, nuclear receptor E75 protein, vacuolar ATP synthase subunit B L form, inositol 1,4,5-trisphosphate receptor, arginine kinase, etc., amounting to 33 differentially modulated proteins were identified successfully. According to Gene Ontology annotation, the identified proteins were classified into nine categories, consisting of immune related proteins, stimulus response proteins, proteins involved in glucose metabolic process, cytoskeleton proteins, DNA or protein binding proteins, proteins involved in steroid hormone mediated signal pathway, ATP synthases, proteins involved in transmembrane transport and ungrouped proteins. Meanwhile, the expression profiles of three up-regulated proteins (SUMO, heat shock protein 70, and arginine kinase) and one down-regulated protein (prophenoloxidase) were further analyzed by real-time RT-PCR at the transcription level after WSSV infection. The results showed that SUMO and heat shock protein 70 were significantly up-regulated at each sampling time point, while arginine kinase was significantly up-regulated at 12 and 24 hpi. In contrast, prophenoloxidase was significantly down-regulated at each sampling time point. The results of this work provided preliminary data on proteins in shrimp hemocytes involved in WSSV infection.

Figure 1. Detection of WSSV in hemocytes of F. chinensis post WSSV infection.

Lane M, marker; Lane 1–2, the first-step PCR products of positive control and negative control; Lane 3–8, the first-step PCR products of hemocytes at 0, 6, 12, 24, 36 and 48 hpi; Lane 9–10, the second-step PCR products of positive control and negative control; Lane 11–16, the second-step PCR products of hemocytes at 0, 6, 12, 24, 36 and 48 hpi.

Figure 2. Representative 2D gel maps of hemocytes proteins of F. chinensis at 24 hpi.

(A) Control hemocytes, (B) WSSV-infected hemocytes, (C) Differentially expressed proteins in a 2D gel. The horizontal axis of the gels was the isoelectric focusing dimension, which stretched in the range of pH 3–10 in a non-linear fashion; the vertical axis was the polyacrylamide gel (12.5%) dimension. Spots were visualized by CBB-G250 staining. Differentially expressed protein spots were circled and labeled with numbers in Fig. 2. C, which correspond to the numbers present in Table S1.

Figure 3. Functional classification of proteins identified in hemocytes of F. chinensis.

A total of 33 identified proteins were categorized into nine groups, and the percentage contribution of each category was calculated.

Figure 4. Expression profiles of four genes in hemocytes of F. chinensis post WSSV infection.

The 18S rRNA was used for normalization of PCR reactions. The bar graphs represent the relative fold changes at each time point post infection, together with error bars which represent mean ± standard deviation (n = 3). Different letters indicates significant difference between groups (p<0.05). (A) SUMO, (B) heat shock protein 70, (C) arginine kinase, (D) prophenoloxidase.