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i(Ehrlichia canis) 120-KDa immunodominant antigenic protein and gene

A technology for Ehrlichia and antigens, used in the production of recombinant proteins, preparations for rickettsial diseases and Ehrlichia, in the field of recombinant proteins, which can solve the problems of cloning and characterizing genes without immunoreactivity

Inactive Publication Date: 2001-11-07
RES DEVMENT FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] A deficiency of the prior art is that the immunoreactive genes for E. canis have not been cloned and characterized
Additionally, the prior art is also deficient in that there are no recombinant proteins for these E. canis immunoreactive genes

Method used

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  • i(Ehrlichia canis) 120-KDa immunodominant antigenic protein and gene
  • i(Ehrlichia canis) 120-KDa immunodominant antigenic protein and gene
  • i(Ehrlichia canis) 120-KDa immunodominant antigenic protein and gene

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Embodiment 1

[0100] Ehrlichia

[0101]The Oklahoma strain of E. canis was provided by Dr. Jacqueline Dawson (Centers of Disease Control, Atlanta, GA). Dr. Edward B. Breitschwerdt (College of Veterinary Medicine, North Calorina State University, Raleigh, NC) provided the Florida strain of E. canis, and three North Carolina isolates (Demon, DJ, and Jake). Dr. R. E. Corstvet (Louisiana State University, Baton Rouge, Louisiana) provided the Louisiana strain of E. canis. Ehrlichia were grown in DH82 cells (a canine macrophage-like cell line, Dawson JE, 1991). When 100% of the cells were infected with Ehrlichia, the DH82 cells were collected with a cell scraper. Centrifuge cells at 17,400xg for 20 minutes. The pellet was pulverized twice on ice with a Braun-Sonic2000 ultrasonic generator at 40W. Cell lysates were added to a step gradient of 42%-36%-30% renografin and centrifuged at 80,000 xg for 60 minutes. Ehrlichia in heavy and light bands were collected (Weiss E, 1975) and washed with ...

Embodiment 2

[0103] DNA preparation

[0104] Genomic DNA of E. canis was prepared from renografin density gradient purified E. canis using the IsoQuick Nucleic Acid Extraction Kit (ORCA Research Inc., Bothell, WA) according to the manufacturer's instructions. Plasmid DNA was purified using a high-purity plasmid isolation kit (Boehringer Mannheim Corp., Indianapolis, IN). PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN Inc., Santa Clarita, CA).

Embodiment 3

[0106] PCR Amplification of 120kDa Protein Gene of Ehrlichia canis

[0107] Primers (Yu, X-J1996) were designed based on the DNA sequence of the E. chaffeensis 120-kDa protein gene (SEQ ID NO: 1-6, FIG. 1 ). Using PCR, the E. canis 120 kDa protein gene was amplified through 30 rounds of 94°C for 30 seconds, 52°C for 1 minute and 72°C for 2 minutes. The PCR amplified product was cloned into pCR2.1 TA cloning vector (Invitrogen, Carlsbad, CA).

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Abstract

The present invention provides a 120-kDa protein gene of Ehrlichia canis, amplified by PCR using primers derived from the DNA sequences flanking the Ehrlichia chaffeensis 120-kDa protein gene. The recombinant E. canis 120-kDa protein contains 14 tandem repeat units with 36 amino acids each. The repeat units are hydrophilic and predicted to be surface-exposed. Also disclosed is that the recombinant E. canis 120-kDa protein is antigenic and reacts with sera from dogs convalescent from canine ehrlichiosis.

Description

Background of the invention [0001] field of invention [0002] The present invention relates generally to the field of molecular biology of parasitic bacteria, particularly Rickettsia-type diseases and preparations of Ehrlichia. More specifically, the present invention relates to the molecular cloning and characterization of the E. canis 120-kDa immunoreactive protein gene. [0003] Description of related fields [0004] Ehrlichia subsp. is an obligate intracellular gram-negative bacterium that resides in the endosomes of hematopoietic cells and infects a variety of animal hosts, including humans, domestic and wild canines, deer, horses, sheep , cattle and wild rodents, etc. Each member of the ehrlichia family has its own specific target cell tropism. Most species of Ehrlichia are monocytotropic (E. canis, E. chaffeensis, E. sennetsu) , Reese (E.risticii) and mouse Ehrlichia (E.muris)) or granulocyte tropism (human granulocyte E. phagocytophila and E. ewingii), in add...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K38/00A61K39/00A61K39/02A61K48/00A61P31/04A61P37/04C07K14/195C07K14/29C07K16/12C12N1/15C12N1/19C12N1/21C12N5/10C12N15/31C12N15/85C12N15/86C12P21/08C12R1/01
CPCC07K14/29A61K39/00A61P31/04A61P37/04Y02A50/30C12N15/11
Inventor D·H·沃克于学杰
Owner RES DEVMENT FOUND
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