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Title: Biochemical, immunological and genetic characterisation of the major outer membrane protein from an ovine abortion strain of Chlamydia psittaci
Author: Tan, Tin Wee
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1989
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Ovine enzootic abortion (OEA) is an economically important disease of ewes caused by a type of Chlamydia psittaci. Vaccines have been available to control this disease for more than 30 years. In the past decade, vaccine efficacy has been poor despite efforts to improve the inactivated whole organism vaccine currently in use. The initial aim of this project was to characterise the antigenic structure of OEA C.psittaci and to identify potentially immunoprotective antigens. Retrospective analyses of sera taken from ewes of different immune status were carried out using immunoblotting. A single 39 to 40 kDa antigen, thought to be the major outer membrane protein (MOMP) of OEA C.psittaci, was implicated as an important immunogen. A series of biochemical analyses including gel electrophoresis, immunoblotting, peptide mapping, surface radio-iodination, detergent fractionation, in vitro oligomerisation and protein microsequencing, as well as electron microscopy, showed conclusively that this antigen was indeed the MOMP of OEA C.psittaci, the analogue of well-characterised MOMPs of other chlamydial strains. OEA MOMP was found to be a major surface-exposed component of the outer membrane fraction of the chlamydial elementary body (EB), and appeared as fine ultrastructural particles (3 to 4 nm in diameter) densely packed on the outermost surface of chlamydial EBs. It possessed epitopes cross-reactive with other chlamydial MOMPs and is a site of heterogeneity between ovine C.psittaci types. Its solubility was enhanced in the presence of reducing agent and MOMP monomers formed disulphide cross-linked oligomers under non-reducing conditions in vitro. It was found to possess an N-terminus identical to C.trachomatis MOMP indicating that cleavage of the signal sequence occurs at the same site to produce a mature protein of 39.5kDa. A method was adapted to isolate MOMP by detergent extraction. An outer membrane fraction, highly enriched in MOMP, was prepared in sufficient quantities for a vaccination-challenge experiment. The results showed that both purified whole elementary bodies and the outer membrane preparation, given in a single dose, could protect ewes from infection and abortion. To test the hypothesis that MOMP was a protective component in these vaccine preparations, sufficient quantities of purified MOMP were needed. A recombinant DNA approach was taken. Firstly, the MOMP gene from a vaccine strain, S26/3, was completely sequenced and extensively analysed in order to develop good strategies of expressing recombinant MOMP (rMOMP). This monocistronic gene contained putative tandem promoter and rho-independent terminator sequences flanking a 1,167 base-pair open reading frame. Comparison with other MOMP gene sequences revealed four highly variable domains interdigitating five constant domains. Using the polymerase chain reaction and specially designed primers, a specific sequence corresponding to the mature MOMP was amplified, cloned into M13 mptac18 viral expression vector and subcloned into three plasmid vectors, pUC8, pRIT5 and pRIT2T, for expression. Several rMOMPs representing the complete mature MOMP and a truncated MOMP were successfully expressed in Escherichia coli. Finally, further analyses showed that these rMOMPs retained antigenic properties of MOMP and that large quantities could be obtained in a highly purified form. Such rMOMPs can now be tested for the presence of epitopes which can protect pregnant ewes from ovine enzootic abortion.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available