Korean J Infect Dis.  1998 Aug;30(4):332-341.

Cloning, Overexpression and Purification of the Heat Shock Protein Hsp58, a Common Antigen of Leptospira Species

• Kim MJ
• Park SC
• Park SH
• Ahn BY

Abstract

BACKGROUND
Humoral immune response is essential for resistance against leptospirosis, and the antigens in-volved in the response might be responsible for pathoge-nesis, diagnosis or immunity. The aim of this study is to clone, overexpress and purify one of leptospiral pro-tein antigens recognized by patients' sera and elucidate it as a diagnostic antigen.
METHODS
We isolated a reactive clone LIA5 from a genomic library of Leptosp ira interrogans serovar lai which expressed a 62 kDa protein co-migrated to a predominant protein of Leptospira by immunoblotting with patients' sera. The sequence of the clone with 2.7 kb leptospiral DNA was determined. The 62 kDa protein encoded by an open reading frame with 1,637 bp was overexpressed in E. coli by pT7 expression vector, and purified to homogeneity by Ni-affinity resin and further electroelution. Patients' sera were examined for IgM or IgG antibodies reacting with the recombinant antigen by strip immunoblot.
RESULTS
Sequence analysis of the clone identified two genes homologous to the hsp58 and hsp 10 of Lepto-sp ira serovar cop enhageni, homologues of the E. coli heat shock protein genes groEL and groES, respectively. The Hsp58 protein was detected commonly in several leptospires by immunoblotting with both patients' sera and rabbit anti-Hsp58 antibody raised against the purified recombinant Hsp58 protein(rHsp58). The predominant class of reacting antibody against rHsp58 in in patients' sera was IgG .
CONCLUSION
The results indicate that Hsp58 is a highly conserved, dominant antigen in humoral immune response to leptospirosis. Further studies are needed to test the rHSP as an immunodiagnostic antigen.

Keyword

Leptospira; Recombinant Hsp58; Overexpression; Purification

MeSH Terms

Antibodies
Clone Cells*
Cloning, Organism*
Diagnosis
DNA
Genomic Library
Heat-Shock Proteins*
Hot Temperature*
Immunity, Humoral
Immunoblotting
Immunoglobulin G
Immunoglobulin M
Leptospira*
Leptospirosis
Open Reading Frames
Sequence Analysis
Antibodies
DNA
Heat-Shock Proteins
Immunoglobulin G
Immunoglobulin M