Cloning and Expression of Shrimp Defense Proteins
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Date
2008-05-10
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Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar
Abstract
Shrimp 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.
Description
Ph.D. Thesis
Keywords
developmental stages, fertilizers, planting, vermicomposting, irrigation, growing media, roses, biofertilizers, grading, yields