APPROACHES TO THE OBJECTIVES
Objective - 1: To study variation in TLR gene expression induced by three types of pathogens / ligands in phylogenetically divergent fishes viz. carp, catfish and shark.
- Design of degenerate PCR primers from the conserved TLRs sequences of zebrafish /pufferfish/catfish.
- Analysis of tissue specific TLRs expression through semi-quantitative RT-PCR/real time quantitative PCR
- Alternatively, preparation of cDNA library for the identification and characterization of TLR -ESTs to generate specific primers for TLR-expression studies.
- Collection of fish tissue – gills, intestine, head kidney, skin and liver from shark,catfish and carps to extract the total RNA and preparation of cDNA.
- Design of PCR primers from available TLR sequences of heterologous species and attempts to analyze the tissue specific expression by semi-quantitative RT-PCR/Real-time quantitative PCR.
Objective - 2: Assessing the divergence of key TLR genes playing a role in the innate immunity of fishes
- Amplification, cloning and sequencing of partial/full length sequence of highly expressed TLR gene(s).
- Employing the sequence data to determine the existence of divergence among the fish species.
- Cloning and sequencing of differentially expressed TLR-genes.
- Full length/ligand binding regions of differentially expressed TLR-genes.
- Analysis of the phylogenetic divergence in the TLR genes of carps, catfish and shark.
Objective - 3: To understand the contribution of TLR signalling pathway(s) in the innate immunity of fishes
- Development of stable cell lines with over-expression the key TLR-genes of homologous/heterologous species
- Activation of TLRs with specific ligands (LPS, LA, CpG and polyI: C) to determine the involvement of downstream effector molecules and their induced interferon and cytokine profiles.
- Preparation of plasmid expression constructs of key TLR-genes.
- Development of stable cell lines over expressing key TLRs of homologous and heterologous species (fish TLR in animal cell and vice versa).
- TLR signalling response to specific ligands (LPS, LA, CpG and poly I: C).
- Testing the TLR-expression plasmid into fish/animal as DNA vaccine.
- Assessment of TLR-ligands induced interferon and cytokines profiles
Objective - 4: Strategic delivery of TLR - ligands and testing their efficacy to modulate innate immunity in-vivo
- Effective delivery of the specific TLR-ligands in a model fish species-Labeo rohita.
- Assesment of innate immune profiles, and their contribution in enhancing the innate immunity.
- Trial on different methods (injection, immersion, feed additive) of delivering TLR ligands into Labeo rohita
- Assessing the duration of the activated TLR and their induced cytokines and interferons.