- INTRODUCTION
- MATERIALS AND METHODS
- Bacteria and vector plasmid.
- Extraction of Ptan strain
- Construction and identification of recombinant plasmid.
- Expression and identification of the truncated Ptan strain 56-kDa protein.
- Purification of Ptan r56.
- Refolding of Ptan r56.
- Western blot.
- Serum samples for ELISA.
- ELISA.
- Human serum samples for IFA and CIA.
- Indirect IFA assay.
- Preparation of diagnostic antigen.
- CIA for detecting total sera anti-O. tsutsugamushi antibodies.
- CIA for detection of anti-O. tsutsugamushi IgG and IgM.
- RESULTS
- DISCUSSION
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
Cao M, Guo H, Tang T, Wang C, Li X, Pan X, Tang J, 2006. Spring scrub typhus, People’s Republic of China. Emerg Infect Disease 12 :1463–1465.
-
2
Amanok K, Suzuki N, Fujita M, Nakamura Y, Suto T, 1993. Serological reactivity of sera from scrub typhus patients against Weil-Felix test antigens. Microbiol Immunol 37 :927–933.
-
3
Tamura A, Ohashi N, Koyama Y, Fukuhara M, Kawamori F, Otsuru M, Wu PF, Lin SY, 1997. Characterization of Orientia tsutsugamushi isolated in Taiwan by immunofluorescence and restriction fragment length polymorphism analyses. Microbiol Lett 150 :225–231.
-
4
Weddle JR, Chan TC, Thompson K, Paxton H, Kelly DJ, Dasch G, Strickman D, 1995. Effectiveness of a dot-blot immunoassay of anti-Rickettsia tsutsugamushi antibodies for serologic analysis of scrub typhus. Am J Trop Med Hyg 53 :43–46.
-
5
Kelly DJ, Wang PW, Gan E, Lewis GE Jr, 1988. Comparative evaluation of the indirect immunoperoxidase test for the se-rodiagnosis of rickettsial disease. Am J Trop Med Hyg 38 :400–406.
-
6
Kim I-S, Seong S-Y, Woo S-G, Choi M-S, Kang J-S, Chang W-H, 1993. High- level expression of a 56-kilodalton protein gene (bor56) of Rickettsia tsutsugamushi Boryong and its application to enzyme-linked immunosorbent assays. J Clin Microbiol 31 :598–605.
-
7
Kawamori F, Akiyama M, Sugieda M, Kanda T, Akahane S, Yamamoto S, Ohashi N, Tamura A, 1993. Two-step polymerase chain reaction for diagnosis of scrub typhus and identification of antigenic variants of Rickettsia tsutsugamushi. J Vet Med Sci 55 :749–755.
-
8
Sugita Y, Nagatani T, Okuda K, Yoshida Y, Makajima H, 1992. Diagnosis of typhus infection with Rickettsia tsutsugamushi by polymerase chain reaction. J Med Microbiol 37 :357–360.
-
9
Singhsilarak T, Leowattana W, Looareesuwan S, Wongchotigul V, Jiang J, Richards AL, Watt G, 2005. Detection of Orientia tsutsugamushi in clinical samples by quantitative real-time polymerase chain reaction. Am J Trop Med Hyg 72 :640–641.
-
10
Cao M, Guo HB, Li YX, Zhang JH, Tang JQ, Tao KH, 2003. The development of the method of detecting Ot DNA using gene chip technique. Chin J Zoonoses 19 :19–22.
-
11
Stover CK, Marana DP, Carter JM, Roe BA, Mardis E, Oaks EV, 1990. The 56-kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning and sequence analysis of the sta 56 gene and precise identification of a strain-specific epitope. J Infect Immun 58 :2076–2084.
-
12
Ohashi N, Nashimoto H, Ikedea H, Tamura A, 1992. Diversity of immunodominant 56-kDa type-specific antigen (TSA) of Rick-ettsia tsutsugamushi. J Biol Chem 267 :12728–12735.
-
13
Cao M, Guo HB, Yu XM, Wang BR, Yang WF, Tang JQ, 2004. Expression, purification and identification of 56 kDa protein of Orientia tsutsugamushi in prokaryotic cells. Chin J Public Health 20 :769–771.
-
14
Kim I-S, Seong S-Y, Woo S-G, Choi M-S, Chang W-H, 1993. Rapid diagnosis of scrub typhus by a passive hemagglutination assay using recombinant 56-kilodalton polypeptides. J Clin Microbial 8 :2057–2060.
-
15
Ching WM, Wang H, Eamsila C, Kelly DJ, Dasch GA, 1998. Expression and refolding of truncated recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi and its use in enzyme-linked immunosorbent assays. Clin Diagn Lab Immunol 5 :519–526.
-
16
Ching WM, Rowland D, Zhang Z, Bourgeois AL, Kelly D, Dasch GA, Devine PL, 2001. Early diagnosis of scrub typhus with a rapid flow assay using recombinant major outer membrane protein antigen (r56) of Rickettsia tsutsugamushi. Clin Diagn Lab Immunol 8 :409–414.
-
17
Chen WJ, Niu DS, Zhang XY, Chen ML, Cui H, Wei WJ, Wen BH, Chen XR, 2003. Recombinant 56-kilodalton major outer membrane protein antigen of Orientia tsutsugamushi Shanxi and its antigenicity. Infect Immun 71 :4772–4779.
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PREPARATION OF RECOMBINANT ANTIGEN OF O. TSUTSUGAMUSHI PTAN STRAIN AND DEVELOPMENT OF RAPID DIAGNOSTIC REAGENT FOR SCRUB TYPHUS
MIN CAO
MIN CAO
Department of Epidemiology and Microbiology, Research Institute for Medicine of Nanjing Command, Jiangsu, China; School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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HENGBIN GUO
HENGBIN GUO
Department of Epidemiology and Microbiology, Research Institute for Medicine of Nanjing Command, Jiangsu, China; School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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TANG TANG
TANG TANG
Department of Epidemiology and Microbiology, Research Institute for Medicine of Nanjing Command, Jiangsu, China; School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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CHANGJUN WANG
CHANGJUN WANG
Department of Epidemiology and Microbiology, Research Institute for Medicine of Nanjing Command, Jiangsu, China; School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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XIANFU LI
XIANFU LI
Department of Epidemiology and Microbiology, Research Institute for Medicine of Nanjing Command, Jiangsu, China; School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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XIUZHEN PAN
XIUZHEN PAN
Department of Epidemiology and Microbiology, Research Institute for Medicine of Nanjing Command, Jiangsu, China; School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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ZHU JIN
ZHU JIN
Department of Epidemiology and Microbiology, Research Institute for Medicine of Nanjing Command, Jiangsu, China; School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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JIAQI TANG
JIAQI TANG
Department of Epidemiology and Microbiology, Research Institute for Medicine of Nanjing Command, Jiangsu, China; School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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Spring scrub typhus has frequently occurred in Pingtan Island, China, since 2000. In this study, we amplified a 1352-bp DNA fragment encoding a truncated 56-kDa outer membrane protein of the Ptan strain, which was isolated from a serum sample of a patient with spring scrub typhus, and cloned it into the pET28a vector for expression. The expression product was a recombinant polypeptide containing a His-tag to facilitate purification on a Ni2+ chromatography column. The recombinant protein was further identified by Western blotting and enzyme-linked immunosorbent assay (ELISA) and appeared to be a good diagnostic antigen candidate. A rapid colloidal gold immunochromatographic assay (CIA) for detecting serum total antibodies, IgG and IgM, which are anti-Orientia tsutsugamushi, was developed, using a mixture of the r56 of the Gilliam and Ptan strains as the diagnostic antigen. CIA performance was tested on a panel of 112 control sera from confirmed cases of scrub typhus. The detection sensitivities of CIA against anti-O. tsutsugamushi total antibodies, IgM, and IgG were 98.2%, 81.2%, and 94.6%, respectively, while that of IFA (using the lysate of the O. tsutsugamushi Gilliam-infected chicken yolk sac as the antigen) against IgG was 85.7%. One hundred five serum samples from healthy individuals and patients with other febrile diseases were tested with CIA as negative controls. Specificities of CIA against anti-O. tsutsugamushi total antibodies, IgM, and IgG were 98.1%, 100%, and 98.9%, respectively, while the specificity of IFA against IgG was 98.9%. These results indicated that CIA was a good assay and could substitute for conventional immunofluorescence assays for diagnosis of scrub typhus.
Author Notes
- INTRODUCTION
- MATERIALS AND METHODS
- Bacteria and vector plasmid.
- Extraction of Ptan strain
- Construction and identification of recombinant plasmid.
- Expression and identification of the truncated Ptan strain 56-kDa protein.
- Purification of Ptan r56.
- Refolding of Ptan r56.
- Western blot.
- Serum samples for ELISA.
- ELISA.
- Human serum samples for IFA and CIA.
- Indirect IFA assay.
- Preparation of diagnostic antigen.
- CIA for detecting total sera anti-O. tsutsugamushi antibodies.
- CIA for detection of anti-O. tsutsugamushi IgG and IgM.
- RESULTS
- DISCUSSION
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
Cao M, Guo H, Tang T, Wang C, Li X, Pan X, Tang J, 2006. Spring scrub typhus, People’s Republic of China. Emerg Infect Disease 12 :1463–1465.
-
2
Amanok K, Suzuki N, Fujita M, Nakamura Y, Suto T, 1993. Serological reactivity of sera from scrub typhus patients against Weil-Felix test antigens. Microbiol Immunol 37 :927–933.
-
3
Tamura A, Ohashi N, Koyama Y, Fukuhara M, Kawamori F, Otsuru M, Wu PF, Lin SY, 1997. Characterization of Orientia tsutsugamushi isolated in Taiwan by immunofluorescence and restriction fragment length polymorphism analyses. Microbiol Lett 150 :225–231.
-
4
Weddle JR, Chan TC, Thompson K, Paxton H, Kelly DJ, Dasch G, Strickman D, 1995. Effectiveness of a dot-blot immunoassay of anti-Rickettsia tsutsugamushi antibodies for serologic analysis of scrub typhus. Am J Trop Med Hyg 53 :43–46.
-
5
Kelly DJ, Wang PW, Gan E, Lewis GE Jr, 1988. Comparative evaluation of the indirect immunoperoxidase test for the se-rodiagnosis of rickettsial disease. Am J Trop Med Hyg 38 :400–406.
-
6
Kim I-S, Seong S-Y, Woo S-G, Choi M-S, Kang J-S, Chang W-H, 1993. High- level expression of a 56-kilodalton protein gene (bor56) of Rickettsia tsutsugamushi Boryong and its application to enzyme-linked immunosorbent assays. J Clin Microbiol 31 :598–605.
-
7
Kawamori F, Akiyama M, Sugieda M, Kanda T, Akahane S, Yamamoto S, Ohashi N, Tamura A, 1993. Two-step polymerase chain reaction for diagnosis of scrub typhus and identification of antigenic variants of Rickettsia tsutsugamushi. J Vet Med Sci 55 :749–755.
-
8
Sugita Y, Nagatani T, Okuda K, Yoshida Y, Makajima H, 1992. Diagnosis of typhus infection with Rickettsia tsutsugamushi by polymerase chain reaction. J Med Microbiol 37 :357–360.
-
9
Singhsilarak T, Leowattana W, Looareesuwan S, Wongchotigul V, Jiang J, Richards AL, Watt G, 2005. Detection of Orientia tsutsugamushi in clinical samples by quantitative real-time polymerase chain reaction. Am J Trop Med Hyg 72 :640–641.
-
10
Cao M, Guo HB, Li YX, Zhang JH, Tang JQ, Tao KH, 2003. The development of the method of detecting Ot DNA using gene chip technique. Chin J Zoonoses 19 :19–22.
-
11
Stover CK, Marana DP, Carter JM, Roe BA, Mardis E, Oaks EV, 1990. The 56-kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning and sequence analysis of the sta 56 gene and precise identification of a strain-specific epitope. J Infect Immun 58 :2076–2084.
-
12
Ohashi N, Nashimoto H, Ikedea H, Tamura A, 1992. Diversity of immunodominant 56-kDa type-specific antigen (TSA) of Rick-ettsia tsutsugamushi. J Biol Chem 267 :12728–12735.
-
13
Cao M, Guo HB, Yu XM, Wang BR, Yang WF, Tang JQ, 2004. Expression, purification and identification of 56 kDa protein of Orientia tsutsugamushi in prokaryotic cells. Chin J Public Health 20 :769–771.
-
14
Kim I-S, Seong S-Y, Woo S-G, Choi M-S, Chang W-H, 1993. Rapid diagnosis of scrub typhus by a passive hemagglutination assay using recombinant 56-kilodalton polypeptides. J Clin Microbial 8 :2057–2060.
-
15
Ching WM, Wang H, Eamsila C, Kelly DJ, Dasch GA, 1998. Expression and refolding of truncated recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi and its use in enzyme-linked immunosorbent assays. Clin Diagn Lab Immunol 5 :519–526.
-
16
Ching WM, Rowland D, Zhang Z, Bourgeois AL, Kelly D, Dasch GA, Devine PL, 2001. Early diagnosis of scrub typhus with a rapid flow assay using recombinant major outer membrane protein antigen (r56) of Rickettsia tsutsugamushi. Clin Diagn Lab Immunol 8 :409–414.
-
17
Chen WJ, Niu DS, Zhang XY, Chen ML, Cui H, Wei WJ, Wen BH, Chen XR, 2003. Recombinant 56-kilodalton major outer membrane protein antigen of Orientia tsutsugamushi Shanxi and its antigenicity. Infect Immun 71 :4772–4779.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 134 | 69 | 4 |
| Full Text Views | 268 | 9 | 0 |
| PDF Downloads | 97 | 12 | 0 |