Molecular and immunological characterisation of Acanthocheilonema viteae chitinase

by Tachu, Babila Julius

Abstract (Summary)
Chitinases are enzymes that break down chitin to its monomers. They are important molecules in the life cycle of parasitic nematodes representing important drug and vaccine targets. Here, three chitinase sequences (I, II and III) were obtained by characterising nine clones from a genomic library of Acanthocheilonema viteae. Southern blots confirmed the existence of three chitinase genes in A. viteae. The organisation of all three genomic sequences is similar, with the greatest difference occurring in exons encoding the serine-threonine rich domain of chitinases: this domain is about ten times larger in sequence III compared to I, but is absent in sequence II. Sequence I and III had features of regularly transcribed genes: start ATG, open reading frame, stop codon and polyadenylation signal. Sequence II lacked the first exon with start ATG. Screening of cDNA libraries from adult female A. viteae worms and L3 (third-stage larvae), as well as reverse transcriptase PCR (RT-PCR) ! showed transcripts in uterine microfilariae, L3 and L4 (fourth-stage larvae) for gene I only. The N-terminal fragment of A. viteae chitinase was cloned into an expression vector and expressed in Escherichia coli. About 80% of the expressed chitinase were found in inclusion bodies and were purified under denaturing conditions. The soluble fraction was about 20% and could be purified under native conditions. Chitinase purified from inclusion bodies was 13-fold less active compared to soluble chitinase. Synthetic peptides (P1 and P2) were designed from the active site of A. viteae chitinase, and used in parallel with chitinase from inclusion bodies in vaccination experiments using the A. viteae / Meriones unguiculatus filariasis model. Vaccination with recombinant protein led to a 29 % significant reduction in adult worm burden in a single experiment. Vaccination with P1 and P2 led to an overall non significant reduction in adult worm burden in two independent experiments. In the P1 group, there was a consistent reduction (39%, p>0.05 and 45%, p<0.05) in mf load that attained significance only in one experiment. In the P2 group, there was no reduction in mf burden in the first experiment, but a significant reduction (75%, p<0.05) in the second. These results suggest that filarial chitinases are potential targets for transmission blocking drugs and vaccines
This document abstract is also available in German.
Bibliographical Information:


School:Humboldt-Universität zu Berlin

School Location:Germany

Source Type:Master's Thesis

Keywords:Acanthocheilonema viteae Chitinase Gene genes Immunisation WD 5075


Date of Publication:08/17/2006

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