Affiliation:
1. Department of Vaccines, National Public Health Institute
2. Haartman Institute, Department of Virology, University of Helsinki
3. HUCH Laboratory Diagnostics, Division of Virology, Helsinki, Finland
Abstract
ABSTRACT
Due to intracellular growth requirements, large-scale cultures of chlamydiae and purification of its proteins are difficult and laborious. To overcome these problems we produced chlamydial proteins in a heterologous host,
Bacillus subtilis
, a gram-positive nonpathogenic bacterium. The genes of
Chlamydia pneumoniae
major outer membrane protein (MOMP), the cysteine-rich outer membrane protein (Omp2), and the heat shock protein (Hsp60) were amplified by PCR, and the PCR products were cloned into expression vectors containing a promoter, a ribosome binding site, and a truncated signal sequence of the α-amylase gene from
Bacillus amyloliquefaciens. C. pneumoniae
genes were readily expressed in
B. subtilis
under the control of the α-amylase promoter. The recombinant proteins MOMP and Hsp60 were purified from the bacterial lysate with the aid of the carboxy-terminal histidine hexamer tag by affinity chromatography. The Omp2 was separated as an insoluble fraction after 8 M urea treatment. The purified proteins were successfully used as immunogens and as antigens in serological assays and in a lymphoproliferation test. The Omp2 and Hsp60 antigens were readily recognized by the antibodies appearing after pulmonary infection following intranasal inoculation of
C. pneumoniae
in mice. Also, splenocytes collected from mice immunized with MOMP or Hsp60 proteins proliferated in response to in vitro stimulation with the corresponding proteins.
Publisher
American Society for Microbiology
Subject
Microbiology (medical),Clinical Biochemistry,Immunology,Immunology and Allergy
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