Division of Virology, IVRI Mukteswar, Nainital 263138, Uttarakhand, India.
The detection and destruction of the invading pathogen is the most important task undertaken by the immune system. Detection of pathogens is carried out by a class of innate immune sensor molecules termed pattern recognition receptors, or PRRs. Several PRRs have been identified each detecting microbial components and initiating responses that program anti-pathogen gene expression profiles and promote adaptive immune responses. Toll like receptors (TLRs) are the major components of the PRR system that detects invading pathogens through conserved molecular structures known as Pathogen Associated Molecular Patterns (PAMP). The recognition of specific PAMP triggers expression of genes, whose product control innate immune response and also instructs further development of the adaptive immune response. To know this pathogen recognition, a comprehensive study is necessary. TLRs have been identified in the farm and companion animals; however, there is no information available on the TLRs of Mithun and their evolutionary lineages with other animal species. The current study is designed to explore the genetics of TLRs of Mithun with evolutionary lineage analysis with other animal species. Mithun is recognized as a religious and very important animal for livestock purpose in North East region of our country. In the present study, the TLR4, which recognizes the Gram negative bacterial Lipopolysaccharide (LPS) has been studied. For the purpose blood was collected from Mithun of Aizawal, Mizoram. The peripheral blood mononuclear cells were isolated using ficoll density gradient centrifugation and stimulated with nonspecific mitogen (ConA) followed by genomic DNA isolation. The PCR conditions were optimized for full length amplification of the TLR4 gene using self designed primers with two overlapping fragment covering the complete ORF of the TLR 4 gene. The individual amplicon was purified from the gel, cloned into pJET1.2 blunt cloning vector; insert release was confirmed by restriction digestion and colony PCR. The positive clones were sequenced. The TLR4 ORF was recognized as 2526bp size. The nucleotide and deduced amino acid sequence analysis with cognate genes of other bovine species revealed that Mithun shows more than 99% sequence homology with Bos taurus, and 94.9%94.6% with other ovine and bovine species at nucleotide level. Amino acid similarity revealed similar picture with 96.1%-94.6% with bovine and 94.9% with ovine. Protein domain architecture as revealed by SMART analysis showed conserved extracellular domain (LRR) and intracellular domain (TIR). Bootstrap phylogenetic analysis by Neighbor-joining method at the amino acid level clustered Mithun more closely with Bos taurus and other bovine species. Work is in progress to find out the corelation between expressions of TLR genes after stimulation with Gram negative bacterial ligands in resistant versus susceptible animal species.
