Cloning and Expression of Shrimp Defense Proteins

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dc.contributor.advisorIndrani Karunasagar
dc.contributor.advisorVenugopal, M.N.
dc.contributor.advisorMaragal, M.M.
dc.contributor.advisorShamsunder, B. A.
dc.contributor.advisorKrishna Bhatt, C.H.
dc.contributor.authorAnuj Tyagi
dc.date.accessioned2016-07-04T14:29:09Z
dc.date.available2016-07-04T14:29:09Z
dc.date.issued2008-05-10
dc.descriptionPh.D. Thesisen_US
dc.description.abstractShrimp aquaculture is affected by various bacterial and viral diseases. In shrimp, immune responses to microbial invasion are mediated by cellular and humoral defense factors. Cellular reactions involve phagocytosis, nodule formation and encapsulation, while humoral reactions involve the prophenoloxidase activating cascade and immune related proteins such as lysozymes, lectins and antimicrobial peptides. These defense protein/enzymes play an important role in elimination of pathogen and prevention of infection related stress. However, during prolonged immune responses, more and more biosynthesis of the proteins is required for continuing the defense or restoring the hemocyte defense proteins, leading to the changes in gene expression as the response progresses. The present study was to characterize a few defense protein of shrimp and to ascertain gene expression in response to microbial challenge. In this study, four defense proteins of shrimp, lysozyme, ferritin, Histone H2A and intracellular fatty acid-binding proteins, were expressed in prokaryotic expression system and their activity characterized. Genes coding for these proteins were amplified by RTPCR and cloned in prokaryotic expression vector. Recombinant clones were sequenced and the nucleotide sequences of all four genes were submitted to GenBank. Recombinant proteins were purified by affinity chromatography. These purified recombinant proteins were used to characterize their in vitro activity. P. monodon lysozyme was a 20. 7 kDa protein belonging to c-type lysozyme. Bacteriolytic activity of lysozyme against different bacterial cultures was determined by solid phase as well as turbiditimetric assay. Lysis was obtained against a broad range of Gram positive and Gram negative bacteria. Minimal inhibitory concentration (MIC) of purified shrimp lysozyme for Vibrio harveyi was 0.47 μg/ml and for Micrococcus luteus, (MIC) 0.12 μg/ml. The lysozyme was found effective against V. harveyi when tested in sea water with and without EDTA. Recombinant ferritin was found to be a 21.8kDa protein, which showed similarity wit L-type ferritins. The predicted 3D structure of the protein showed the presence of 6 α helix with amino acid residues involved in iron (III) binding (Giu54, Asp57, Glu58, Glu61). Cleavage site and iron uptake activity of recombinant ferritin was determined. The purified recombinant ferritin helped in reducing the mortality and offered protection to shrimps when challenged with Vibrio harveyi. Recombinant histone H2A was found to be 16 kDa protein with 47 – 98% similarity to H2A protein and H2A derived antimicrobial peptides of other organisms. However, the recombinant protein and its pepsin treated fraction did not show any antimicrobial activity by solid phase assay. The intracellular fatty acid binding protein (IFA) was found to be a 18kDa protein Clustal generated multiple sequence alignment of deduced amino acid sequence showed 48 – 100% similarity with fatty acid binding proteins (FABPs) and cellular retinoic acid-binding proteins (CRABPs) of various arthropods. Essential amino acid triad of R/R/Y, considered important for binding of all-trans retinoic acid in vertebrates was also present in P. monodon IFA (Arg110, Arg130, Tyr132). Construction of phylogenetic tree by neighbor-joining method revealed that P. monodon IFA formed a separate cluster with other arthropod’s FABPs and CRSBPs and more closely related to human FABPs than human CRABP Gene expression in shrimp in response to WSSV challenge was studied by suppression subtractive hybridization (SSH). mRNA obtained from hemocytes of P. monodon challenged with WSSV and from control animals was used in this assay, which would help identification of genes differentially expressed in response to WSSV infection. Fragments of differentially expressed genes were cloned and sequenced. Various bioinformatics tools were used to analyze the nucleotide sequences and to ascertain their similarity to sequences in GenBank database. A total of 50 differentially expressed sequences were discovered in our SSH cDNA library. Out of these 50 sequences, 8 were similar to previously discovered genes in P. monodon. These included gene coding for a protein (Rab7) that binds to WSSV envelop protein, VP28, β-glucan binding protein of P. monodon, C-type lectin of P. monodon, NADH dehydrogenase of P. monodon, caspase of P. monodon, glutathione peroxidase of L. vannamei, Thrombospondin of Penaeus monodon and microsatellite sequence of P. monodon. 20 sequences showed similarity with homologous genes of various other organisms, reported first time in penaeid shrimp. These included proteins such as protienases involved in immune function, enzymes involved in metabolic functions and transport proteins. Rest of the sequences did not show any similarity to sequences in GenBank database.en_US
dc.identifier.urihttp://krishikosh.egranth.ac.in/handle/1/68412
dc.language.isoenen_US
dc.publisherKarnataka Veterinary, Animal and Fisheries Sciences University, Bidaren_US
dc.subFishery Microbiology
dc.subjectdevelopmental stages, fertilizers, planting, vermicomposting, irrigation, growing media, roses, biofertilizers, grading, yieldsen_US
dc.these.typePh.D
dc.titleCloning and Expression of Shrimp Defense Proteinsen_US
dc.typeThesisen_US
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