酪氨酸磷酸化蛋白和胞质动力蛋白在对虾白斑症病毒wssv感染过程中的作用研究-study on the role of tyrosine phosphorylated protein and cytoplasmic dynamic protein in wssv infection of prawn white spot disease virus.docxVIP

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酪氨酸磷酸化蛋白和胞质动力蛋白在对虾白斑症病毒wssv感染过程中的作用研究-study on the role of tyrosine phosphorylated protein and cytoplasmic dynamic protein in wssv infection of prawn white spot disease virus.docx

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酪氨酸磷酸化蛋白和胞质动力蛋白在对虾白斑症病毒wssv感染过程中的作用研究-study on the role of tyrosine phosphorylated protein and cytoplasmic dynamic protein in wssv infection of prawn white spot disease virus

万方数据万方数据 two peaks at 1 and 12 hour post infection. In present work, the change of protein tyrosine phosphorylation in shrimp hemocytes was investigated by FCM, western blotting and confocal microscopy during WSSV infection. All the results showed that the level of protein tyrosine phosphorylation was significantly up-regulated with a similar pattern of fluctuation. Confocal microscopic observation revealed that the tyrosine phosphorylated proteins were localized in nucleus as well as cytoplasm, and five tyrosine phosphorylated proteins were detected in hemocytes by western blotting using phosphotyrosine-specific antibody, which were identified to be histone H2A, histone H3, histone H4, alpha-2-macroglobulin (A2M) and heat shock protein 70 (HSP70) by mass spectrometry. Moreover, the expression levels of h2a, h3 and h4 were examined to be significantly up-regulated post WSSV infection by quantitative real-time RT-PCR. The full-length cDNA of cytoplasmic dynein intermediate chain (FcDYNCI) was first cloned from the hemocyte of Fenneropenaeus chinensis, which consists of 2582 bp and encodes a polypeptide of 660 amino acids (Aa). The predicted protein molecular mass was 73.40 kDa, and the estimated isoelectric point was 5.16. Multiple sequence alignment showed that FcDYNCI shared high identities with DYNC1I2 from four species in Insecta. These suggested that FcDYNCI was a member of cytoplasmic dynein 1 family and might be clustered into isoform 2. The FcDYNCI mRNA was most highly expressed in hemocytes, which was significantly up-regulated post WSSV infection. At 12 h post infection (hpi), confocal microscopic observation showed that WSSV virions could be co-localized with the cytoplasmic dynein in hemocytes. After silencing by specific FcDYNCI dsRNA, the FcDYNCI mRNA level and the protein amount of FcDYNCI in hemocytes both exhibited a significant reduction, and the expression levels of three WSSV genes ie1, wsv477 and vp28 all exhibited the greatest decreases at 24 h