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Arockiaraj J.,University of Malaya | Easwvaran S.,University of Malaya | Vanaraja P.,University of Malaya | Singh A.,Center for Aquaculture Research and Extension | And 2 more authors.
Molecular Biology Reports | Year: 2012

The prophenoloxidase activating system is an important innate immune response against microbial infections in invertebrates. The major enzyme, phenoloxi-dase, is synthesized as an inactive precursor and its activation to an active enzyme is mediated by a cascade of clip domain serine proteinases. In this study, a cDNA encoding a prophenoloxidase activating enzyme-III from the giant freshwater prawn Macrobrachium rosenbergii, designated as MrProAE-III, was identified and characterized. The full-length cDNA contains an open reading frame of 1110 base pair (bp) encoding a predicted protein of 370 amino acids including an 22 amino acid signal peptide. The MrProAE-III protein exhibits a characteristic sequence structure of a long serine proteases-trypsin domain and an N- and C-terminal serine proteases-trypsin family histidine active sites, respectively, which together are the characteristics of the clip-serin proteases. Sequence analysis showed that MrProAE-III exhibited the highest amino acid sequence similarity (63%) to a ProAE-III from Atlantic blue crab, Callinectes sapidus. MrProAE-III mRNA and enzyme activity of MrProAE-III were detectable in all examined tissues, including hepatopancreas, hemocytes, pleopods, walking legs, eye stalk, gill, stomach, intestine, brain and muscle with the highest level of both in hepatopancreas. This is regulated after systemic infectious hypodermal and hematopoietic necrosis virus infection supporting that it is an immune-responsive gene. These results indicate that MrProAE-III functions in the proPO system and is an important component in the prawn immune system. © 2011 Springer Science+Business Media B.V.


Arockiaraj J.,University of Malaya | Easwvaran S.,University of Malaya | Vanaraja P.,University of Malaya | Singh A.,Center for Aquaculture Research and Extension | And 2 more authors.
Fish and Shellfish Immunology | Year: 2012

In this study, we reported a full length of catalase gene (designated as MrCat), identified from the transcriptome database of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrCat is 2504 base pairs in length, and encodes 516 amino acids. The MrCat protein contains three domains such as catalase 1 (catalase proximal heme-ligand signature) at 350-358, catalase 2 (catalase proximal active site signature) at 60-76 and catalase 3 (catalase family profile) at 20-499. The mRNA expressions of MrCat in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction (qRT-PCR). The MrCat is highly expressed in digestive tract and all the other tissues (walking leg, gills, muscle, hemocyte, hepatopancreas, pleopods, brain and eye stalk) of M. rosenbergii taken for analysis. The expression is strongly up-regulated in digestive tract after IHHNV challenge. To understand its biological activity, the recombinant MrCat gene was constructed and expressed in Escherichia coli BL21 (DE3). The recombinant MrCat existed in high thermal stability and broad spectrum of pH, which showed over 95% enzyme activity between pH 5 and 10.5, and was stable from 40 °C to 70 °C, and exhibited 85-100% enzyme activity from 30 °C to 40 °C. © 2012 Elsevier Ltd.


Arockiaraj J.,University of Malaya | Easwvaran S.,University of Malaya | Vanaraja P.,University of Malaya | Singh A.,Center for Aquaculture Research and Extension | And 2 more authors.
Fish and Shellfish Immunology | Year: 2012

Caspase 3c (MrCasp3c) was sequenced from the freshwater giant prawn Macrobrachium rosenbergii using Illumina Solexa Genome Analyzer Technique. MrCasp3c consisted of 2080 bp nucleotide encoded 521 polypeptide with an estimated molecular mass of 59 kDa. MrCasp3c sequence contains caspase family p20 domain profile and caspase family p10 domain profile at 236-367 and 378-468 respectively. The quantitative real time PCR analysis revealed a broad expression of MrCasp3c with the highest expression in haemocyte and the lowest in stomach. The expression of MrCasp3c after challenge with the infectious hypodermal and haematopoietic necrosis virus (IHHNV) was tested in haemocyte. In addition, MrCasp3c was expressed in Escherichia coli by prokaryotic expression plasmid pMAL-c2x. The enzyme activity of MrCasp3c was also found to be up-regulated by IHHNV in haemocyte and hepatopancreas tissues. This study suggested that MrCasp3c may be an effector caspase associated with the induction of apoptosis which is potentially involved in the immune defence of M. rosenbergii. © 2011 Elsevier Ltd.


Arockiaraj J.,University of Malaya | Easwvaran S.,University of Malaya | Vanaraja P.,University of Malaya | Singh A.,Center for Aquaculture Research and Extension | And 2 more authors.
Fish and Shellfish Immunology | Year: 2012

In this study, we have reported a full length of peroxiredoxin (designated MrPrdx) gene, identified from the transcriptome of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrPrdx is 940 base pairs in length, and encodes 186 amino acids. MrPrdx contains a long thioredoxin domain in the amino acid sequence between 34 and 186. The gene expressions of MrPrdx in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction. MrPrdx is highly expressed in all the other tissues of M. rosenbergii considered for analysis and the highest in gills. The expression is strongly up-regulated in gills after IHHNV infection. To understand MrPrdx functional properties, the recombinant MrPrdx protein was expressed in Escherichia coli BL21 (DE3) and purified. A peroxidise activity assay was conducted using recombinant MrPrdx protein at different concentrations. This peroxidises activity showed that the recombinant MrPrdx is a thiol-dependant protein. Additionally, this result showed that recombinant MrPrdx protein, as a secretory protein can remove H 2O 2 and protect DNA damage. This finding leads a possible way to propose the recombinant MrPrdx protein as an effective medicine for reactive oxygen species (ROS) related diseases. © 2012 Elsevier Ltd.


Arockiaraj J.,SRM University | Avin F.A.,University of Malaya | Vanaraja P.,University of Malaya | Easwvaran S.,University of Malaya | And 3 more authors.
Fish and Shellfish Immunology | Year: 2012

NF kappa B inhibitor alpha (MrNFκBI-α) was sequenced from a freshwater prawn Macrobrachium rosenbergii. The MrNFκBI-α protein contains a long ankyrin repeat region circular domain between 193 and 413 along with its 6 repeats (ankyrin repeat 1,2,3,4,5 and 6). An IκB degradation motif and a putative PEST motif is present at 37-64 and 418-471 of the N- and C-terminal regions of MrNFκBI-α respectively. The gene expressions of MrNFκBI-α in healthy and infectious hematopoietic and hypodermal necrosis virus (IHHNV), poly I:C, Aeromonas hydrophila and Enterococcus faecium injected M. rosenbergii were examined using quantitative real time PCR. The MrNFκBI-α is expressed in all the tissues taken for examination and the highest is observed in hemocytes. The MrNFκBI-α gene expression is strongly up-regulated in hemocytes of prawn after IHHNV, poly I:C, A. hydrophila and E. faecium infection. This result indicates an important role of MrNFκBI-α in M. rosenbergii immune system. This, however, remains to be verified by further studies. © 2012 Elsevier Ltd.

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