Cloning, Overexpression and Purification of the Heat Shock Protein Hsp58, a Common Antigen of Leptospira Species.
- Author:
Min Ja KIM
1
;
Seung Chul PARK
;
Se Hoon PARK
;
Byung Yoon AHN
Author Information
1. Department of Internal Medicine, College of Medicine, Korea Univers ity, Seoul, Korea.
- Publication Type:Original Article
- Keywords:
Leptospira;
Recombinant Hsp58;
Overexpression;
Purification
- MeSH:
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
- From:Korean Journal of Infectious Diseases
1998;30(4):332-341
- CountryRepublic of Korea
- Language:Korean
-
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.
