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Ehrlichia chaffeensis 28 kDa outer membrane protein multigene family

Inactive Publication Date: 2002-05-30
RES DEVMENT FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The prior art is deficient in the lack of the knowledge of many of the sequences of the genes in the p28 multigene family of E. chaffeensis.

Method used

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  • Ehrlichia chaffeensis 28 kDa outer membrane protein multigene family
  • Ehrlichia chaffeensis 28 kDa outer membrane protein multigene family
  • Ehrlichia chaffeensis 28 kDa outer membrane protein multigene family

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example 2

[0060] PCR Amplification of the p28 Multigene Locus

[0061] Ehrlichia chaffeensis genomic DNA was prepared by using an IsoQuick Nucleic Acid Extraction Kit (ORCA Research Inc., Bothell, Wash.) according to the instructions of the manufacturer. The unknown sequences of the p28 multigene locus were amplified by PCR using the Universal GenomeWalker Kit (Clontech Laboratories, Inc., Palo Alto, Calif.). Briefly, the E. chaffeensis genomic DNA was digested respectively with Dra I, EcoR V, Pvu II, Sca I, and Stu I. The enzymes were chosen because they generated blunt ended DNA fragments to ligate with the blunt-end of the adapter. The digested E. chaffeensis genomic DNA fragments were ligated with a GenomeWalker Adapter, which had one blunt end and one end with 5' overhang. The ligation mixture of the adapter and E. chaffeensis genomic DNA fragments was used as template for PCR. Initially, the p28 gene-specific primer amplified the known DNA sequence and extended into the unknown adjacent ge...

example 4

[0064] Gene Analysis

[0065] DNA sequences and deduced amino acid sequences were analyzed using DNASTAR software (DNASTAR, Inc., Madison, Wis.). The signal sequence of the deduced protein was analyzed by using the PSORT program, which predicts the presence of signal sequences (McGeoch, 1985, Von Heijne, 1986) and detects potential transmembrane domains (Klein, 1985). Phylogenetic analysis was performed by the maximum parsimony method of the PAUP 4.0 software (Sunderland Mass.: Sinauer Associates, 1998). Bootstrap values for the consensus tree were based on analysis of 1000 replicates.

example 5

[0066] DNA Sequence Accession Numbers

[0067] The DNA sequences of the E. chaffeensis p28 genes were assigned GenBank accession numbers: AF230642 for the DNA locus of the p28-1 to p28-13 and AF230643 for the DNA locus of p28-20 and p28-21.

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Abstract

The 28-kDa outer membrane proteins (P28) of Ehrlichia chaffeensis are encoded by a multigene family consisting of 21 members located in a 23-kb DNA fragment in the genome of E. chaffeensis. Fifteen of these proteins are claimed herein as novel sequences. The amino acid sequence identity of the various P28 proteins was 20-83%. Six of 10 tested p28 genes were actively transcribed in cell culture grown E. chaffeensis. RT-PCR also indicated that each of the p28 genes was monocistronic. These results suggest that the p28 genes are active genes and encode polymorphic forms of the P28 proteins. The P28s were also divergent among different isolates of E. chaffeensis. The large repertoire of the p28 genes in a single ehrlichial organism and antigenic diversity of the P28 among the isolates of E. chaffeensis suggest that the P28s may be involved in immune avoidance.

Description

[0001] The present invention relates generally to the fields of microbiology, bacteriology and molecular biology. More specifically, the present invention relates to the molecular cloning and characterization of the Ehrlichia chaffeensis 28 kD outer membrane protein multigene family.DESCRIPTION OF THE RELATED ART[0002] Ehrlichia are small, obligatory intracellular, gram negative bacteria which reside in endosomes inside host cells. Ehrlichiae usually cause persistent infection in their natural animal hosts (Andrew and Norval, 1989, Breitschwerdt et al., 1998, Dawson et al., 1994, Dawson and Ewing, 1992, Harrus et al., 1998, Telford et al., 1996). Persistent or prolonged Ehrlichia infections in human hosts have also been documented (Dumler et al., 1993, Dumler and Bakken, 1996, Horowitz, et al., 1998, Roland et al. 1994). The persistent infection may be caused by the antigenic variation of the Ehrlichia omp-2 and p28 outer membrane protein family due to differential expression or rec...

Claims

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Application Information

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IPC IPC(8): A61K39/00C07K14/29C12N1/21
CPCC07K14/29A61K39/00Y02A50/30
Inventor WALKER, DAVID H.YU, XUE-JIE
Owner RES DEVMENT FOUND
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