The first MmmSC display library was constructed by Persson and co

The first MmmSC display library was constructed by Persson and co-workers [16] and more recently, the approach was also applied to Mycoplasma hyopneumoniae [17] as a way of identifying immunogenic polypeptides. To locate genes coding for potentially immunogenic proteins, enzymatically-generated fragments of MmmSC chromosomal DNA were used to construct a genome-specific filamentous phage display library MEK activation which was subjected to selection using antibodies from a CBPP outbreak in Botswana [18] and an experimentally infected animal from Mali designated

C11 [19]. CD4+ T-cell activation and IFNγ release are associated with an IgG2 humoral immune response [20] while IgA is associated with local mucosal immunity. Accordingly, both immunoglobulin

classes were used separately to select peptides as well as using total IgG. Using this approach together with computer algorithms designed to identify linear B-cell epitopes [21], five genes were chosen to be expressed for further analysis and testing to establish whether they were recognised by sera from cattle obtained during a natural outbreak of the disease. Results Construction of a fragmented-genome library A pIII fusion protein phage display library of approximately 4 × 105 primary clones displaying peptides derived from the MmmSC genome was constructed by ligating DNA fragments ranging in size from approximately 30 to 900 bp as determined by agarose gel electrophoresis into

a filamentous phage display vector. The probability of the genome p38 MAPK cancer being represented was 0.97 if the average insert size was 240 bp. DNA sequencing of 16 arbitrarily-chosen clones showed no obvious bias towards any particular region of the mycoplasmal genome. Of the 16, two copies of one of the sequenced DNA inserts were in-frame and in the correct orientation. The largest insert was ZD1839 datasheet 178 base pairs and the smallest 52. To verify that the library was large and diverse enough to identify other unknown MmmSC antigens, it was first screened in a defined model system by panning on immuno-purified IgG prepared from a rabbit immune serum directed against amino acid residues 328-478 of the proline-rich MmmSC glycoprotein which is coded for by ORF5 (EMBL/GenBank accession number CAE77151). Multiple copies of overlapping peptides that mapped to a defined region on the target glycoprotein spanning residues 333 to 445 were selected (Figure 1). Figure 1 Schematic representation showing alignment of the hypothetical proline-rich glycoprotein ORF5 with selected phage fusion peptides. Antigenic regions suggested by the presence of overlapping sequences located between amino acid residues 358-365 and 388-410 are indicated by shading.

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