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Molecular cloning and characterisation of Dermatophilus congolensis serine protease genes

Mine, Ontiretse Madisa (1996) Molecular cloning and characterisation of Dermatophilus congolensis serine protease genes. PhD thesis, Murdoch University.

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Abstract

The bacterium Dermatophilus congolensis causes the skin disease of sheep known as ovine dermatophilosis. Control of ovine dermatophilosis by vaccination in Western Australia has largely been unsuccessful, because of variation in virulence and antigenicity between isolates of the bacterium. A recent study of 30 Australian isolates of D. congolensis showed that the haemolytic activity and production of serine proteases had some link with virulence ranking. Recent work on parasites and other organisms has led to experimental vaccines based on serine proteases secreted by these organisms.

When this work was being planned, preliminary vaccination trials carried out by Dr. T. M. Ellis and colleagues using serine proteases from D. congolensis strains AG (less virulent) and MB (more virulent) were showing that the protease proteins could be incorporated into a vaccine against D. congolensis. The purification of D. congolensis serine proteases using current methods resulted in yields too low for research purposes. The work in this thesis describes the first stage in the application of recombinant DNA technology to the production of a vaccine against ovine dermatophilosis.

A novel strategy using PCR and generic degenerate primers designed from other bacterial serine protease gene sequences, was developed to produce a nucleic acid probe for the D. congolensis serine proteases.

Genomic DNA libraries were constructed in λgtl0 vector for the two strains of D. congolensis AG and MB. The probe was used to isolate the serine protease genes from the libraries. The genes were sequenced and their amino acid sequences predicted. The comparison of the amino acid sequences of D. congolensis strains with those of the other bacterial serine proteases showed similarity which consisted of structurally conserved regions (SCRs). The SCRs consisted of stretches of amino acids with strong sequence homology which occurred around the active site residues and other essential secondary structural elements.

The antigenicity of the amino acid sequences of the gene fragments between the histidine and serine active residues of the D. congolensis strains (AG and MB) was determined using the MacVector computer program. An epitope was predicted and a corresponding peptide synthesised. Preliminary results showed that one of the sheep immunised with serine proteases purified from D. congolensis had antibody to the peptide.

A gene fragment encoding amino acid sequence between the histidine and serine active site residues of the AG strain was directionally cloned into pQE30, pQE31 and pQE32 plasmid vectors. The recombinant plasmids were screened for inserts by restriction digestion of plasmid DNA, and the plasmids carrying the inserts were used to transform E. coli M15[pREP4] cells. The transformants were screened for protein production by induction with IPTG. A clone producing a recombinant protein was isolated and a small amount of the protein purified.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Biological and Environmental Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: repository@murdoch.edu.au. Thank you.
Supervisor(s): Carnegie, Patrick and Ellis, Trevor
URI: http://researchrepository.murdoch.edu.au/id/eprint/53235
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