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VETERINARY IMMUNOLOGY

Identification, Bioinformatics Analyses, and Expression of Immunoreactive Antigens of Mycoplasma haemofelis

Joanne B. Messick, Andrea P. Santos
Joanne B. Messick
Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana
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  • For correspondence: jmessic@purdue.edu
Andrea P. Santos
Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana
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DOI: 10.1128/CVI.05060-11
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  • Fig. 1.
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    Fig. 1.

    Genes identified by immunoscreening of Mycoplasma haemofelis Lambda ZapII libraries. The clones discovered are indicated by the abbreviations listed in Table 1, and the location in the genome of M. haemofelis is shown. The 16S rRNA gene and the gene coding for DnaA (chromosomal replication initiator) are marked in gray as reference points. The scale is in kilobases.

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    Fig. 2.

    SDS-PAGE and Western blot analyses of the putative antigens P6D-orf0908, P10C-orf1238, P10E-orf0279, P10C-orf1239, P21B-orf1546l, and P29-orf0177. (Top) SDS-PAGE showing the expression of putative antigens (SimplyBlue SafeStain; Invitrogen); (middle) Western blot results of the putative antigens against the His6 tag; (bottom) Western blot results of the putative antigens against convalescent pooled plasma from an experimentally infected cat. Lane 1, fragment of the DnaK control (approximately 31.75 kDa); lane 2, fragment of P6D-orf0908 (approximately 18.65 kDa); lane 3, fragment of P10C-orf1238 (approximately 20.52 kDa); lane 4, fragment of P10E-orf0279 (approximately 19.07 kDa); lane 5, fragment of P10C-orf1239 (approximately 18.88 kDa); lane 6, fragment of P21B-orf1546 (approximately 12.56 kDa); lane 7, fragment of P29-orf0177 (approximately 21.71 kDa); lanes M, molecular mass markers (Precision Plus Protein Kaleidoscope; Bio-Rad).

Tables

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  • Table 1.

    Reactive clones by immunoscreening of Mycoplasma haemofelis Lambda ZapII libraries and BLASTx results against nonredundant protein sequence database

    Insert identificationGenBank accession no.Sequence similarity by BLASTx against the MollicutesE value
    P3-orf1165a,bGS928052ACQ84443.1, adhesin (Mycoplasma hyopneumoniae)0.002
    P5-orf1816a,b,cGS928053NP_853366.1, thymidine phosphorylase (M. gallisepticum)0.37
    P6D-orf0908a,b,dGS928054ZP_06610317.1, hypothetical protein MALL_0643 (M. alligatoris)0.44
    P6D-orf0909bGS928055ZP_06610593.1, hypothetical protein MALL_0515 (M. alligatoris)0.63
    P7-orf0259GS928056ACU78513.1, triose-phosphate isomerase (M. mycoides)8E-36
    P7-orf0260GS928057NP_073101.2, phosphoglyceromutase (M. genitalium)9E-128
    P7-orf0261GS928058BAH70152.1, hypothetical protein (M. fermentans)0.026
    P7-orf0262aGS928059NSe
    P7-orf0263aGS928060ABD47695.1, adhesin-like protein P146 (M. hyopneumoniae)1.4
    P7-orf0264GS928061AAZ44718.2, conserved hypothetical protein (M. hyopneumoniae)0.22
    P9D-orf1202GS928062ZP_06610215.1, type I restriction modification DNA protein (M. alligatoris)2E-15
    P9D-orf1203GS928063YP_003303059.1, type I restriction enzyme specificity protein (M. hominis)0.005
    P9D-orf1204GS928064ZP_02931536.1, type I restriction enzyme S protein (Ureaplasma parvum)4E-10
    P9D-orf1205GS928065YP_003303059.1, type I restriction enzyme specificity protein (M. hominis)2E-12
    P10B-orf1747a,bGS928066YP_002284694.1, putative lipoprotein (Ureaplasma urealyticum)3.1
    P10B-orf1748GS928067YP_003515875.1, hypothetical protein MAGa7180 (M. agalactiae)0.17
    P10B-orf1749a,bGS928068NS1.1
    P10B-orf1750GS928069YP_003560289.1, hyaluronoglucosaminidase (M. crocodyli)1.1
    P10C-orf1238a,b,dGS928070YP_002000188.1, massive surface protein MspK (M. arthritidis)0.004
    P10C-orf1239a,b,dGS928071ZP_04563878.1, transcriptional regulator (Mollicutes bacterium D7)0.002
    P10E-orf0279a,dGS928072NP_757929.1, hypothetical protein MYPE5440 (M. penetrans)3E-50
    P10E-orf0280GS928073NP_975564.1, pseudouridylate synthase D (M. mycoides)1E-41
    P15-orf0941GS928074YP_002960937.1, hypothetical protein MCJ_004270 (M. conjunctivae)0.0024
    P15-orf0942GS928075NS
    P15-orf0943bGS928076ACU78785.1, conserved hypothetical protein (M. mycoides)0.28
    P15-orf0944GS928077AAO39838.1, AvgC variable lipoprotein (M. agalactiae)0.17
    P15-orf0945a,bGS928078YP_002000023.1, massive surface protein MspF (M. arthritidis)0.63
    P15-orf0946a,bGS928079ZP_02695921.2, hypothetical protein UUR13 (U. urealyticum)1.8
    P15-orf0947a,bGS928080ZP_06610731.1, conserved hypothetical protein (M. alligatoris)0.37
    P15-orf0948bGS928081YP_279005.1, lysyl-tRNA synthetase (M. hyopneumoniae)0.63
    P15-orf0949bGS928082NP_758309.1, phenylalanyl-tRNA synthetase subunit beta (M. penetrans)0.48
    P17A-orf1526GS928083CAB62239.1, P75 protein (M. hominis)0.28
    P17A-orf1527a,bGS928084ZP_04564868.1, conserved hypothetical protein (Mollicutes bacterium D7)0.002
    P17A-orf1528bGS928085ZP_02971377.1, conserved hypothetical protein (U. parvum)0.37
    P17A-orf1529bGS928086YP_016078.1, hypothetical protein MMOB3810 (M. mobile)0.37
    P18-orf0127GS928087NP_757967.1, hypoxanthine-guanine phosphoribosyltransferase (M. penetrans)6E-07
    P18-orf0128aGS928088YP_002000162.1, hypothetical protein MARTH (M. arthritidis)0.13
    P20/22-orf0326aGS928089NP_757466.1, DNA-directed RNA polymerase subunit beta (M. penetrans)0
    P21A-orf0675a,bGS928090NP_757933.1, translocase (Mycoplasma penetrans)3.1
    P21B-orf1544a,bGS928091YP_002000128.1, massive surface protein MspH (M. arthritidis)0.044
    P21B-orf1545GS928092NP_757749.1, hypothetical protein MYPE3620 (M. penetrans)0.057
    P21B-orf1546a,b,dGS928093YP_001256183.1, hypothetical protein MAG_0390 (M. agalactiae)0.097
    P21B-orf1547bGS928094NP_975173.1, hypothetical protein MSC_0170 (M. mycoides)1.1
    P21B-orf1548bGS928095YP_001799373.1, hypothetical protein PAa (C. Phytoplasma australiense)0.13
    P24-orf1679aGS928096YP_002000015.1, massive surface protein MspC (M. arthritidis)1.13
    P24-orf1680GS928097NS
    P24-orf1681bGS928098NP_758083.1, hypothetical protein MYPE6950 (M. penetrans)0.28
    P26-orf0285GS928099NP_853008.2, translation elongation factor Tu (EF-Tu) (M. gallisepticum)8E-129
    P26-orf0286GS928100NP_757969.1, adenylosuccinate synthetase (M. penetrans)4E-110
    P26-orf0287GS928101ADC31594.1, ribosomal biogenesis GTPase (M. gallisepticum)2E-31
    P27A-orf1350bGS928102YP_002000022.1, massive surface protein MspE (M. arthritidis)2E-03
    P28-orf1521a,bGS928103YP_002961131.1, hypothetical protein MCJ_006330 (M. conjunctivae)0.130
    P28-orf1522bGS928104YP_016010.1, SWF/SNF family helicase (M. mobile)0.630
    P28-orf1523bGS928105ZP_04563267.1, conserved hypothetical protein (Mollicutes bacterium D7)0.220
    P29-orf0175GS928106YP_001621362.1, ketose bisphosphate aldolase (Acholeplasma laidlawii)5E-102
    P29-orf0176GS928107NP_072863.1, cochaperone GrpE (M. genitalium)4E-22
    P29-orf0177aGS928108NP_758284.1, heat shock protein DnaJ (M. penetrans)2E-69
    P29-orf0178GS928109NP_109915.1, elongation factor G (M. pneumoniae)0
    P32C-orf0088GS928110ZP_03079605.1, arginyl-tRNA synthetase (U. urealyticum)7E-75
    P33B-orf1097GS928111YP_002000188.1, massive surface protein MspK (M. arthritidis)0.057
    • ↵a Putative proteins selected by bioinformatics analyses to be cloned and expressed by the Gateway system.

    • ↵b Putative proteins predicted to have a transmembrane domain(s) by hidden Markov model and dense alignment surface methods.

    • ↵c Shading indicates ORFs within the same plaque.

    • ↵d Putative proteins that were reactive against immune plasma and negative against nonimmune plasma by Western blotting.

    • ↵e NS, no significant similarity found.

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Identification, Bioinformatics Analyses, and Expression of Immunoreactive Antigens of Mycoplasma haemofelis
Joanne B. Messick, Andrea P. Santos
Clinical and Vaccine Immunology Aug 2011, 18 (8) 1275-1281; DOI: 10.1128/CVI.05060-11

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Identification, Bioinformatics Analyses, and Expression of Immunoreactive Antigens of Mycoplasma haemofelis
Joanne B. Messick, Andrea P. Santos
Clinical and Vaccine Immunology Aug 2011, 18 (8) 1275-1281; DOI: 10.1128/CVI.05060-11
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    • ABSTRACT
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