Proteomic analysis of protein expression in Penaeus vannamei hemocytes upon taura syndrome virus infection

การวิเคราะห์การแสดงออกของโปรตีนในเม็ดเลือดแดงของกุ้งขาวซึ่งติดเชื้อไวรัสทอร่าซอนโดรมในเชิงโปรติโอม

Name: Phattara-orn Chongsatja

LCSH: Penaeidae

LCSH: Taura syndrome virus

MeSH: Hemocytes

MeSH: Hemocyanin

MeSH: Proteomics

Abstract: Taura Syndrome Virus (TSV) was isolated and partially purified from the hemolymph of infected Penaeus vannamei by using Ficoll® density gradients. TSV was detected in infected hemocytes after 4 hours post-infection by RT-PCR amplification. The detected quantity increased with longer times of infection, indicating propagation and accumulation of virus in the infected shrimp. To understand the molecular responses of crustacean hemocytes to TSV infection, the study applied a gel-based proteomics approach to investigate changes in expression levels of hemocytic proteins in P. vannamei upon TSV infection. At 24 hours post-infection, quantitative intensity analysis of 2-D gel and nanoLC-ESIMS/ MS revealed that 11 forms of 8 proteins were significantly up-regulated, whereas 9 forms of 5 proteins were significantly down-regulated in the infected shrimp. These altered proteins are involved in host defense mechanisms (hemocyanin, catalase, carboxylesterase, transglutaminase and gluthatione transferase), signal transduction (14-3-3 zeta), carbohydrate metabolism (acetylglucosamine pyrophosphorylase), cellular structure and integrity (betatubulin, beta-actin, tropomyosin and myosin), and ER-stress response (protein disulfide isomerase). Semi-quantitative RT-PCR and Western blot analysis confirmed the up-regulation of 14-3-3 at both mRNA and protein levels. Interestingly, several altered protein spots were identified as fragments of hemocyanin. Mass spectrometric analysis of the hemocyanin spots present at the more acidic region identified peptides covering residues only at the C-terminus of hemocyanin. In contrast, the hemocyanin spots present at the less acidic area had sequence coverage only at the N-terminus of hemocyanin. Bioinformatic calculation clearly confirmed that theoretical pI of a C-terminal hemocyanin fragment was more acidic (spot #7; pI = 4.81) than that of an N-terminal hemocyanin fragment (spot #15; pI = 5.64). As 3/4 of C-terminal fragments and 2/3 of N-terminal hemocyanin fragments were up- and down-regulated, respectively, these two terminal fragments could have different functional roles in a viral infected shrimp

Abstract: Taura Syndrome Virus (TSV) was isolated and partially purified from the hemolymph of infected Penaeus vannamei by using Ficoll® density gradients. TSV was detected in infected hemocytes after 4 hours post-infection by RT-PCR amplification. The detected quantity increased with longer times of infection, indicating propagation and accumulation of virus in the infected shrimp. To understand the molecular responses of crustacean hemocytes to TSV infection, the study applied a gel-based proteomics approach to investigate changes in expression levels of hemocytic proteins in P. vannamei upon TSV infection. At 24 hours post-infection, quantitative intensity analysis of 2-D gel and nanoLC-ESIMS/ MS revealed that 11 forms of 8 proteins were significantly up-regulated, whereas 9 forms of 5 proteins were significantly down-regulated in the infected shrimp. These altered proteins are involved in host defense mechanisms (hemocyanin, catalase, carboxylesterase, transglutaminase and gluthatione transferase), signal transduction (14-3-3 zeta), carbohydrate metabolism (acetylglucosamine pyrophosphorylase), cellular structure and integrity (betatubulin, beta-actin, tropomyosin and myosin), and ER-stress response (protein disulfide isomerase). Semi-quantitative RT-PCR and Western blot analysis confirmed the up-regulation of 14-3-3 at both mRNA and protein levels. Interestingly, several altered protein spots were identified as fragments of hemocyanin. Mass spectrometric analysis of the hemocyanin spots present at the more acidic region identified peptides covering residues only at the C-terminus of hemocyanin. In contrast, the hemocyanin spots present at the less acidic area had sequence coverage only at the N-terminus of hemocyanin. Bioinformatic calculation clearly confirmed that theoretical pI of a C-terminal hemocyanin fragment was more acidic (spot #7; pI = 4.81) than that of an N-terminal hemocyanin fragment (spot #15; pI = 5.64). As 3/4 of C-terminal fragments and 2/3 of N-terminal hemocyanin fragments were up- and down-regulated, respectively, these two terminal fragments could have different functional roles in a viral infected shrimp.

Mahidol University

Address: NAKHON PATHOM

Email: liwww@mahidol.ac.th

Name: Chartchai Krittanai

Role: Thesis Advisors

Created: 2007

Modified: 2553-06-21

Issued: 2010-06-11

วิทยานิพนธ์/Thesis

application/pdf

CallNumber: TH P536p 2007

eng

DegreeName: Doctor of Philosophy

Level: Doctoral Degree

Descipline: Molecular Genetics and Genetic Engineering

Grantor: Mahidol University

©copyrights Mahidol University

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