Abstract
The coccidioidal T27K vaccine is protective in mice against respiratory challenge with Coccidioides posadasii (C. posadasii) arthroconidia. The vaccine is a subcellular multicomponent preparation that has not been fully characterized. To identify potential protective antigens in the heterogeneous mixture, the vaccine has been separated by two-dimensional gel electrophoresis and then analyzed for seroreactive proteins using immunoblot analysis with pooled sera from patients with coccidioidomycosis. Two seroreactive spots of identical apparent molecular weight were identified and sequenced using tandem mass spectrometry. Three peptides were generated, two of which matched a tentative consensus sequence in the TIGR C. posadasii 2.0 gene index database that is similar to fungal 1,2-α-mannosidases. The 5′ and 3′ ends of the mannosidase cDNA were mapped using rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR), and a full-length cDNA was then obtained using reverse-transcription (RT) PCR. The cDNA was cloned and sequenced and expressed as a recombinant protein. The predicted protein consists of 519 amino acids, has a theoretical molecular weight and pI of 56,918 Da and 4.84, respectively, and is very similar (>60%) to other fungal 1,2-α- mannosidases. Class I 1,2-α-mannosidase enzyme activitywas also detected in the T27K vaccine using the substrate, Man-α-1,2-Man-α-OCH 3 in a spectrophotometric assay.
| Original language | English (US) |
|---|---|
| Title of host publication | Annals of the New York Academy of Sciences |
| Pages | 164-180 |
| Number of pages | 17 |
| Volume | 1111 |
| DOIs | |
| State | Published - Sep 2007 |
Publication series
| Name | Annals of the New York Academy of Sciences |
|---|---|
| Volume | 1111 |
| ISSN (Print) | 00778923 |
| ISSN (Electronic) | 17496632 |
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Keywords
- Coccidioides
- Fungus
- Immunoblot
- Mannosidase
- Mass spectrometry
- Rapid amplification of cDNA ends
- Reverse-transcription polymerase chain reaction
- Seroreactive
- Two-dimensional gel electrophoresis
- Vaccine
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
Cite this
Molecular cloning and expression of a cDNA encoding a Coccidioides posadasii 1,2-α-mannosidase identified in the coccidioidal T27K vaccine by immunoproteomic methods. / Lunetta, Jennine M.; Simmons, Keira A.; Johnson, Suzanne M.; Pappagianis, Demosthenes.
Annals of the New York Academy of Sciences. Vol. 1111 2007. p. 164-180 (Annals of the New York Academy of Sciences; Vol. 1111).Research output: Chapter in Book/Report/Conference proceeding › Conference contribution
}
TY - GEN
T1 - Molecular cloning and expression of a cDNA encoding a Coccidioides posadasii 1,2-α-mannosidase identified in the coccidioidal T27K vaccine by immunoproteomic methods
AU - Lunetta, Jennine M.
AU - Simmons, Keira A.
AU - Johnson, Suzanne M.
AU - Pappagianis, Demosthenes
PY - 2007/9
Y1 - 2007/9
N2 - The coccidioidal T27K vaccine is protective in mice against respiratory challenge with Coccidioides posadasii (C. posadasii) arthroconidia. The vaccine is a subcellular multicomponent preparation that has not been fully characterized. To identify potential protective antigens in the heterogeneous mixture, the vaccine has been separated by two-dimensional gel electrophoresis and then analyzed for seroreactive proteins using immunoblot analysis with pooled sera from patients with coccidioidomycosis. Two seroreactive spots of identical apparent molecular weight were identified and sequenced using tandem mass spectrometry. Three peptides were generated, two of which matched a tentative consensus sequence in the TIGR C. posadasii 2.0 gene index database that is similar to fungal 1,2-α-mannosidases. The 5′ and 3′ ends of the mannosidase cDNA were mapped using rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR), and a full-length cDNA was then obtained using reverse-transcription (RT) PCR. The cDNA was cloned and sequenced and expressed as a recombinant protein. The predicted protein consists of 519 amino acids, has a theoretical molecular weight and pI of 56,918 Da and 4.84, respectively, and is very similar (>60%) to other fungal 1,2-α- mannosidases. Class I 1,2-α-mannosidase enzyme activitywas also detected in the T27K vaccine using the substrate, Man-α-1,2-Man-α-OCH 3 in a spectrophotometric assay.
AB - The coccidioidal T27K vaccine is protective in mice against respiratory challenge with Coccidioides posadasii (C. posadasii) arthroconidia. The vaccine is a subcellular multicomponent preparation that has not been fully characterized. To identify potential protective antigens in the heterogeneous mixture, the vaccine has been separated by two-dimensional gel electrophoresis and then analyzed for seroreactive proteins using immunoblot analysis with pooled sera from patients with coccidioidomycosis. Two seroreactive spots of identical apparent molecular weight were identified and sequenced using tandem mass spectrometry. Three peptides were generated, two of which matched a tentative consensus sequence in the TIGR C. posadasii 2.0 gene index database that is similar to fungal 1,2-α-mannosidases. The 5′ and 3′ ends of the mannosidase cDNA were mapped using rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR), and a full-length cDNA was then obtained using reverse-transcription (RT) PCR. The cDNA was cloned and sequenced and expressed as a recombinant protein. The predicted protein consists of 519 amino acids, has a theoretical molecular weight and pI of 56,918 Da and 4.84, respectively, and is very similar (>60%) to other fungal 1,2-α- mannosidases. Class I 1,2-α-mannosidase enzyme activitywas also detected in the T27K vaccine using the substrate, Man-α-1,2-Man-α-OCH 3 in a spectrophotometric assay.
KW - Coccidioides
KW - Fungus
KW - Immunoblot
KW - Mannosidase
KW - Mass spectrometry
KW - Rapid amplification of cDNA ends
KW - Reverse-transcription polymerase chain reaction
KW - Seroreactive
KW - Two-dimensional gel electrophoresis
KW - Vaccine
UR - http://www.scopus.com/inward/record.url?scp=35348851951&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=35348851951&partnerID=8YFLogxK
U2 - 10.1196/annals.1406.015
DO - 10.1196/annals.1406.015
M3 - Conference contribution
C2 - 17363438
AN - SCOPUS:35348851951
SN - 1573316881
SN - 9781573316880
VL - 1111
T3 - Annals of the New York Academy of Sciences
SP - 164
EP - 180
BT - Annals of the New York Academy of Sciences
ER -