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The exploration of candidate genes for immune response in cattle may be vital for improving our understanding regarding the species specific response to pathogens. Toll-like receptor 4 (TLR4) is mostly involved in protection against the deleterious effects of Gram negative pathogens. Approximately 2.6 kb long cDNA sequence of TLR4 gene covering the entire coding region was characterized in two Indian milk cattle (Vrindavani and Tharparkar). The phylogenetic analysis confirmed that the bovine TLR4 was apparently evolved from an ancestral form that predated the appearance of vertebrates, and it is grouped with buffalo, yak, and mithun TLR4s. Sequence analysis revealed a 2526-nucleotide long open reading frame (ORF) encoding 841 amino acids, similar to other cattle breeds. The calculated molecular weight of the translated ORF was 96144 and 96040.9 Da; the isoelectric point was 6.35 and 6.42 in Vrindavani and Tharparkar cattle, respectively. The Simple Modular Architecture Research Tool (SMART) analysis identified 14 leucine rich repeats (LRR) motifs in bovine TLR4 protein. The deduced TLR4 amino acid sequence of Tharparkar had 4 different substitutions as compared toBos taurus, Sahiwal, and Vrindavani. The signal peptide cleavage site predicted to lie between 16th and 17th amino acid of mature peptide. The transmebrane helix was identified between 635–657 amino acids in the mature peptide. [ABSTRACT FROM AUTHOR]