The deduced amino acid sequences between rRmLTI, EST CK186726, an

The deduced amino acid sequences between rRmLTI, EST CK186726, and BmTI-6 are 99% identical. Nucleic acid sequence coding for six additional amino acids (EAEAEF) in the N-terminus, and thirty two amino acids (VPRAAAAASFLEQKLISEEDLNSAVDHHHHHH) in the C-terminal portion of the putative rRmLTI product was added during cloning procedures to allow insertion of a restriction site and coding sequence for the poly-His peptide. The similarity between

their partial amino acid sequences suggested that RmLTI in larvae is a member of the Kunitz-bovine pancreatic trypsin inhibitor (BPTI) family like BmTI-6 in the ovary of adult female cattle ticks. Further exploration of the putative function of RmLTI is reflected in Fig. 7. Relevant protein signature features identified in the deduced amino acid sequence encoded in the expressed sequence tag CK186726 include three putative Kunitz-BPTI SB431542 concentration domains and two putative Kunitz proteinase inhibitor I2 conserved sites. As noted in BmTI-6, six N-glycosylation

sites were present in the partial protein sequence of RmLTI. Six cysteine residues were observed within each of the three Kunitz domains, which are thought to form disulfide linkages contributing stability to the compact polypeptide in its folded form. Assessment of the efficacy against cattle tick infestation Bax protein in bovines using a vaccine containing the recombinant form of a member of the Kunitz-BPTI family from R. microplus produced

in the P. pastoris expression system is reported for the first time here. A specific and robust humoral immune response against rRmLTI was achieved with the vaccination protocol consisting of three immunizations, each applied every two weeks. The 32% efficacy obtained with the rRmLTI formulation reflects the significant challenge of discovering highly efficacious antigens protecting cattle against R. microplus infestation. Vaccination over experiments where Bos indicus cattle were immunized with a mixture of purified larval trypsin inhibitors containing one or two Kunitz-type domains afforded 72.8% efficacy against R. microplus infestation [22]. In contrast, the level of immunoprotection obtained in crossbred cattle vaccinated with the synthetic polypeptide containing 29 aa residues derived from the N terminus of the R. microplus trypsin inhibitor A was 18.4% [23]. As the gene encoding RmLTI remains to be fully characterized, the apparent discrepancy between specific antibody levels and the low level of efficacy obtained with the rRmLTI vaccine may be due to the partial gene sequence of the EST used to produce the recombinant protein product in the yeast expression system. Alternatively, the structural and functional redundancy in proteins belonging to the Kunitz family present in R.

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