Article Figures & Data
Figures
- FIG. 1.
(A) Deduced amino acid sequence alignment of VirB9 proteins from six members of the family Anaplasmataceae. Aligned identical amino acids are in white letters on a black background. Gaps in the alignment are indicated by dashes. The alignments were generated by the CLUSTAL V method in the MegAlign program (DNASTAR). Conserved sequences are indicated by arrows. The GenBank accession numbers of the VirB9 proteins of E. chaffeensis ArkansasT, A. phagocytophilum HZ, Wolbachia sp. strain wTai, and Wolbachia sp. strain wKueYo are AF392617 , AF392618 , AB045234 , and AB045235 , respectively. The sequence of N. sennetsu MiyayamaT is available at http://www.tigr.org . (B) Phylogram based on the deduced amino acid sequences of the virB9 genes of E. canis and similarities with selected gram-negative bacteria. The amino acid sequences were aligned by the CLUSTAL V method, and phylogenic analysis was performed with the use of the PHYLIP software package (version 3.5). The order Rickettsiales is boxed. The GenBank accession numbers of the virB9 sequences of the organisms used in this analysis are as follows: Agrobacterium tumefaciens Ti, J03320 ; Bartonella henselae, AF182718 ; Bordetella pertussis ptl, D47301 ; Brucella abortus, AF226278 ; Brucella suis, AF141604 ; Legionella pneumophila lvh, Y19029 ; Rickettsia prowazekii, AJ235271 ; Rickettsia conorii, NC_003103 .
- FIG. 2.
virB9 expression by E. canis in naturally infected R. sanguineus tick groups from Arizona and in samples of dog blood. Total RNA was extracted and subjected to reverse transcription-PCR, as described in Materials and Methods. The amplified products were separated on agarose gels and visualized with ethidium bromide. Lane M, molecular size marker (1 Kb Plus DNA ladder; Invitrogen); lane PC, product amplified by PCR with DNA from E. canis culture as a template and respective primer pairs, used as a positive control for PCR and as a determinant of amplicon size on the gel; lane NT, no template (negative control). Samples were run in the presence (+) and absence (−) of reverse transcriptase, as a control for DNA contamination. DH82, uninfected DH82 cells. (A) Weekly expression levels of virB9 between day 0 and 6 weeks after infection of dog PBMCs (A1, Arizona-2; A2, New Mexico-2); (B) virB9 expression in dog C (OklahomaT, virB9 expression was analyzed only at the third week after infection in dog C); (C) virB9 expression in culture of E. canis VDE and OklahomaT in DH82 cells; (D) virB9 expression in naturally infected tick groups (R12, one engorged female; R13, two engorged females and one unengorged male; R24, one male and one semiengorged female; R48, two males and one semiengorged female; R56, two unengorged females; and R17 [E. canis 16S rRNA-negative ticks as a control], two engorged females and one male).
- FIG. 3.
(A) SDS-polyacrylamide gel electrophoresis analysis of purified E. canis OklahomaT rVirB9. Affinity-purified (AP) rVirB9 (2 μg) was subjected to SDS-polyacrylamide gel electrophoresis followed by Coomassie blue staining. M, molecular size marker. The numbers on the left are molecular masses, in kilodaltons. (B) Western immunoblot analysis to detect E. canis VirB9-specific antibody in plasma samples from infected dogs. pET33b-transformed E. coli was used as a negative control. rVirB9, affinity-purified recombinant fusion protein of E. canis. Antigens (15 μg of E. coli and 2 μg of rVirB9) were subjected to Western blot analysis with serum derived from the blood of various infected dogs (OK, Oklahoma; OH, Ohio; AZ, Arizona; NM, New Mexico). The arrow on the right indicates the apparent molecular mass of rVirB9, based on broad-range prestained standards (Bio-Rad Laboratories, Richmond, Calif.).
Tables
- TABLE 1.
virB9 primer pairs used in this study
Primer Sequence Purpose EcavB9f1 5′-GAATTTACCCATCCAAGTGGC-3′ Cloning and sequencing EcavB9r2 5′-CCAACAACGTTTACATTCTCTTC-3′ EcavB9f3 5′-TCGGGATCCGAGCATAGCTGATAA CCCTGTTAC-3′ Cloning and expression EcavB9r3 5′-AGTGCGGCCGCTTATAGTTTTTTGT TAGTTATTTTCACAG-3′ EcavB9of 5′-CATTATCATTTCAATACGTAACTC-3′ Cloning and sequencing EcavB9or 5′-TTTTGATTTTCTTCTGACATAGTG-3′ EcavB9f 5′-GCACACTCCATAAGCATAGCTG-3′ Expression analysis EcavB9r 5′-CTCTTAGGTCAGAGTACTCGTC-3′
- Top
- Article
- ABSTRACT
- Cloning and sequencing of virB9.
- Sequence analysis of the virB9 gene from different geographic locations.
- Analysis of virB9 expression.
- Expression of recombinant VirB9 (rVirB9) in Escherichia coli and Western blot analysis.
- Nucleotide sequence accession numbers.
- ACKNOWLEDGMENTS
- FOOTNOTES
- REFERENCES
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