Expression and Refolding of Truncated Recombinant Major Outer Membrane Protein Antigen (r56) of Orientia tsutsugamushi and Its Use in Enzyme-Linked Immunosorbent Assays
| Autor: | H. Wang, C. Eamsila, Wei-Mei Ching, Daryl J. Kelly, G. A. Dasch |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Microbiology (medical)
Protein Folding Orientia tsutsugamushi Molecular Sequence Data Clinical Biochemistry Immunology Gene Expression Enzyme-Linked Immunosorbent Assay Scrub typhus Sensitivity and Specificity Article Immunoglobulin G Immunoenzyme Techniques Antigen medicine Animals Humans Immunology and Allergy Amino Acid Sequence DNA Primers Antiserum Antigens Bacterial Base Sequence biology bacterial infections and mycoses biology.organism_classification medicine.disease Antibodies Bacterial Orientia Molecular biology Peptide Fragments Recombinant Proteins Scrub Typhus Genes Bacterial Case-Control Studies biology.protein Rabbits Antibody Indirect immunoperoxidase assay Bacterial Outer Membrane Proteins |
| Zdroj: | Clinical Diagnostic Laboratory Immunology. 5:519-526 |
| ISSN: | 1098-6588 1071-412X |
| DOI: | 10.1128/cdli.5.4.519-526.1998 |
| Popis: | The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. The gene encoding this protein from Karp strain was cloned into the expression vector pET11a. The recombinant protein (r56) was expressed as a truncated nonfusion protein (amino acids 80 to 456 of the open reading frame) which formed an inclusion body when expressed in Escherichia coli BL21. Refolded r56 was purified and compared to purified whole-cell lysate of the Karp strain of O. tsutsugamushi by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for reactivity with rabbit sera prepared against eight antigenic prototypes of O. tsutsugamushi as well as several other species of Rickettsiales and nonrickettsial antigens. Refolded r56 exhibited broad reactivity with the rabbit antisera against the Orientia prototypes, and the ELISA reactions with the r56 and Karp whole-cell lysate antigens correlated well ( r = 0.81, n = 22, sensitivity compared to that of standard ELISA of 91%). Refolded r56 did not react with most antisera against other rickettsial species or control antigens (specificity = 92%, n = 13) using a positive cutoff value determined with eight uninfected rabbit sera. Refolded r56 was evaluated further by ELISA, using 128 sera obtained from patients with suspected scrub typhus from Korat, Thailand, and 74 serum specimens from healthy Thai soldiers. By using the indirect immunoperoxidase assay as the reference assay, the recombinant antigen exhibited a sensitivity and specificity of 93% or greater for detection of both IgG and IgM in the ELISA at 1:400 serum dilution. These results strongly suggest that purified r56 is a suitable candidate for replacing the density gradient-purified, rickettsia-derived, whole-cell antigen currently used in the commercial dipstick assay available in the United States. |
| Databáze: | OpenAIRE |
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