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Cloning, characterization and expression analysis of Nucleoside Phosphorylase 5a (PNP 5a) from Crucian carp (Carassius auratus gibelio)

Cloning, characterization and expression analysis of Nucleos...

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中国水产学会鱼病专业委员会2013年学术研讨会

作者： patarida podok Lijuan Xu Dan Xu Liqun Lu Key Laboratory of Aquatic Genetic Resources of the Ministry of Agriculture Shanghai Ocean University

Purine nucleoside phosphorylase 5a(PNP5a) is a ubiquitous enzyme of purine metabolism,which catalyses the reversible phosphorolysis of purine *** inhibitors,affecting the survival and function of T-lymphocyte are potential candidates antitumour and antiviral *** a study to identify differentially expressed immune-related genes with suppression subtractive hybridization(SSH) technique,PNP5a gene was determined to be significantly up-regulated in kidney tissue of Crucian carp(Carassius auratus gibelio) infected with CyHv-2.A real-time RT-PCR assay was developed to quantitatively examine its tissue *** demonstrated that,PNP5a was expressed at low level in all *** monitor the response of PNP5a to Cyprinid herpesvirus 2 infection or Aeromonas hydrophila infection,the expression level of PNP5a was *** up-regulate expression of PNP5a at 72 and 6 hour were *** is the first report on expression analysis of PNP5a in response to viral and bacterial *** at different time points after pathogen challenge in Crucian carp(Carassius auratus gibelio),that suggest a potential role for innate immunity.

关键词： Purine Nucleoside Phosphorylase 5a(PNP 5a) Crucian Carp(Carassius auratus gibelio) Real-time RT-PCR

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Active transcription of MHC class I and Lysozyme C in response of crucian carp（Carassius auratus gibelio） to Cyprinid herpesvirus 2 infection: implication for their basic role in antiviral innate immunity

Active transcription of MHC class I and Lysozyme C in respon...

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2014年中国水产学会学术年会

作者： patarida podok Lijuan Xu Dan Xu Liqun Lu Key Laboratory of Aquatic Genetic Resources of the Ministry of Agriculture Shanghai Ocean University

MHC class I（MHC I） and Lysozyme C（LYZ C） are play important roles associated with disease resistant in fish species and believed to play in antiviral innate immunity. Through suppression subtractive hybridization（SSH） analysis, we previously identified MHC I and LYZ C as the two genes that were expressed in both of moribund and survivor SSH libraries after Cyprinid herpesvirus 2（Cy HV-2） infections. Here, we have further characterized two genes and their response to viral challenge The open reading frames（ORF） of MHC I and LYZ C were cloned from crucian carp（Carassius auratus gibelio）. The full-length c DNAs of the two genes contained an ORF of 1,038 and 438 bp, encode a polypeptide of 345 and 145 amino acids, respectively. Phylogenetic analysis indicated that MHC I and LYZ C were most closely related to common carp. We found that two genes were expressed at low levels in all of the tissues examined in healthy crucian carp. The m RNA expression of MHC I and LYZ C in crucian carp kidney after Cy HV-2 infection by using real-time RT PCR analysis at different time points, during challenge experiments, the peak of MHC I was observed at 72 hpi.（5.55-fold, P＜0.01）. While, 48 hpi（3.87-fold, P＜0.01） was the highest expression time points for LYZ C. For the fish died and survived from viral infection, expression levels of MHC I were 3.17 fold and 3.05 fold（P＜0.01）, respectively. Same as in the expression levels of LYZ C, were significantly upregulated in both died and survived fish（2.32 fold and 2.56 fold（P＜0.05）, respectively）. These data suggested MHC I and LYZ C were involved in cellular response genes to Cy HV-2 but not play a key role in resistance.

关键词： crucian carp Carassius auratus gibelio innate immunity MHC class I Lysozyme C

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Proteomic identification and characterization of Ctenopharyngodon idella tumor necrosis factor receptor-associated protein 1（CiTrap1）: an anti-apoptosis factor upregulated by grass carp reovirus infection

Proteomic identification and characterization of Ctenopharyn...

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2014年中国水产学会学术年会

作者： Yan Li Yanan Zhang Tu Wang patarida podok Dan Xu Liqun Lu Key Laboratory of Freshwater Fishery Germplasm Resources Ministry of Agriculture Shanghai Ocean University

Human tumor necrosis factor receptor-associated protein 1（Trap1） is a mitochondrial protein identical to heat shock protein 75（HSP75） that plays an important role in protecting cells from oxidative stress and apoptosis. In this study, grass carp（Ctenopharyngodon idella） tumor necrosis factor receptor-associated protein 1（designated as Ci Trap1） was identified through two-dimensional electrophoresis（2-DE） analysis and its patterns of expression was investigated in grass carp kidney（CIK） cells infected with grass carp reovirus（GCRV）. The full length c DNA of Ci Trap1 was cloned and sequenced revealing it to contain an opening reading frame of 2157 bp that encoded a peptide of 718 amino acids. Phylogenetic analyses indicated that the Ci Trap1 shared 87 % homology with the zebrafish（Danio rerio）. The transcriptional level of Ci Trap1 in CIK cells was upregulated 24 h after virus infection with similar expression patterns of the Ci Trap1 gene observed after poly（I:C） stimulation. At later stages of infection the expression level was downregulated. Following virus infection the expression levels of grass carp PTEN-induced putative kinase 1（PINK1） and Sorcin, whose human homologues interact with Trap1 during anti-apoptosis, exhibited similar expression patterns to that of Ci Trap1. Typical characteristics of apoptosis, including the formation of an apoptotic body and DNA fragmentation, were observed in CIK cells infected with GCRV by DAPI staining, DNA ladder electrophoresis and TUNEL assay. Although the m RNA expression level of caspase-3, as well as the Bax/Bcl-2 ratio, was upregulated during GCRV infection, the apoptosis was seemed to be self-limiting by our apoptosis assay. The results of this study suggest that Ci Trap1 is involved in the host’s innate immune response to viral infection, possibly through protecting infected cells from apoptosis.

关键词： Grass carp(Ctenopharyngodon idella) proteomic analysis tumor necrosis factor receptor-associated protein 1 apoptosis grass carp reovirus

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