Cloning and Expression of the 44-Kilodalton Major Outer Membrane Protein Gene of the Human Granulocytic Ehrlichiosis Agent and Application of the Recombinant Protein to Serodiagnosis

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Journal of Clinical Microbiology, June 1998, p. 1666-1673, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Cloning and Expression of the 44-Kilodalton Major Outer Membrane Protein Gene of the Human Granulocytic Ehrlichiosis Agent and Application of the Recombinant Protein to Serodiagnosis

N. Zhi,¹ N. Ohashi,¹ Y. Rikihisa,¹^,^* H. W. Horowitz,² G. P. Wormser,² and K. Hechemy³

Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio,¹ and Division of Infectious Diseases, Westchester County Medical Center, New York Medical College, Valhalla,² and New York State Department of Health, Albany,³ New York

Received 2 January 1998/Returned for modification 19 February 1998/Accepted 17 March 1998

A 44-kDa major outer membrane protein of the human granulocytic ehrlichiosis (HGE) agent is an immunodominant antigen in humaninfection. A gene encoding this protein was cloned and sequenced.Southern blot results revealed the existence of multigenes homologousto the P44 gene in the genome of the HGE agent. The recombinant44-kDa protein (rP44) was expressed by using expression vectorpET30a. The reactivity of the affinity-purified rP44 was evaluatedby Western immunoblot analysis and dot blot immunoassay. Westernimmunoblot analysis showed that mouse anti-rP44 serum reactedwith 44- to 42-kDa proteins in six different HGE agent strainstested except strain 2, in which three proteins of 42, 40, and38 kDa were recognized. Eleven HGE patient serum samples, a horseanti-HGE serum, and a horse anti-Ehrlichia equi serum recognizedthe rP44 protein. This suggests that rP44 is an HGE-E. equi group-specificantigen. Neither human anti-Ehrlichia chaffeensis serum nor rabbitanti-Borrelia burgdorferi serum reacted with rP44. Sera from twopatients coinfected with the HGE agent and B. burgdorferi reactedpositively with rP44 and the HGE agent. Sera from 20 HGE patientswith indirect fluorescent-antibody (IFA) titers ranging from 1:20to 1:2,560 gave distinct positive reactions in a dot immunoblotassay. There was a positive correlation between the color densitiesof the dot reactions and the IFA titers when greater than 50 ngof recombinant antigen per dot was used. The use of the affinity-purifiedrP44 protein as antigen would provide a more specific, consistent,and simpler serodiagnosis for HGE than the use of whole infectedcells or purified HGE agents.

^* Corresponding author. Mailing address: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio StateUniversity, 1925 Coffey Rd., Columbus, OH 43210-1093. Phone: (614)292-9677. Fax: (614) 292-6473. E-mail: rikihisa.1{at}osu.edu.

Journal of Clinical Microbiology, June 1998, p. 1666-1673, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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