CHAPTER 5 Immunological studies
5.2 Production of antibodies in chickens against synthetic peptides
5.2.1 Selection of peptides
The amino acid sequences of the Plasmodium falciparum protein, PFC0760c and the Plasmodium yoelii yoelii protein, PY05757, were analysed for regions predicted to be immunogenic using the Predict 7 program (Cármenes et al., 1989). The algorithms predicting hydrophilicity (Hopp and Woods, 1981; Kyte and Doolittle, 1982), flexibility (Karplus and Shulz, 1985), surface probability (Janin et al., 1978) and antigenicity (Welling et al., 1985) were used for the entire amino acid sequences of PY05757 (Figure 5.1 A) and PFC0760c (Figure 5.2 A). Regions of 12 or more consecutive amino acids having values above zero for hydrophilicity, flexibility, and surface probability were considered sequences possible for peptide synthesis. A number of sequences were predicted to be favourable for peptide synthesis for both proteins. However, many of these sequences produced significant matches during BLAST searches, to other P. yoelii yoelii proteins, mouse (Mus musculus) and chicken (Gallus gallus) proteins (results not shown). It was necessary to select peptide sequences that would be sufficiently foreign to chickens, in order to raise chicken anti-peptide antibodies. It was also essential for the peptide sequence selected for PY05757 to not have sequence similarity with mouse proteins, or other P.
yoelii yoelii proteins, as the antibodies raised would be required to detect the native PY05757 protein in P. yoelii yoelii infected mouse red blood cells. Plasmodium proteins tend to be lysine rich (Flick et al., 2004; Vedadi et al., 2007) and therefore any sequences having multiple lysine residues were also not further considered for synthesis.
The peptide sequence SDDDNRQIQDFE, corresponding to amino acid residues 1341-1352 of PY05757 was identified as a candidate for raising antibodies. The plots of hydrophilicity, flexibility and surface probability were all above zero (Figure 5.1 B). In addition, a BLAST search at NCBI with the “search for short nearly exact matches” option (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) produced a significant match only for the hypothetical protein PY05757 (Table 5.1). No Gallus gallus (chicken) or Mus musculus (mouse) proteins produced significant matches, suggesting that the peptide sequence is likely to be foreign and hence immunogenic in chickens and mice
Due to the small molecular mass of the peptide, it was necessary to couple the peptide to a carrier protein, rabbit albumin, to increase the antibody production. The peptide SDDDNRQIQDFE was synthesized with a cysteine residue at the C-terminus, allowing coupling to the rabbit albumin carrier with 3-maleimidobenzoic acid N- hydroxysuccinimide (MBS) via the sulfhydryl group (Section 2.30). The cysteine residue was synthesised on the C-terminus of the peptide, as Predict 7 predicted the amino acids at the C-terminus to be slightly less immunogenic than those at the N-terminus (Figure 5.1 B).
In addition, peptides FKLGSCYLYIINRNLKEI and CFKLGSCYLYIINRNLKEI were synthesized (GenScript Corporation, USA). These peptides were identical in sequence, except that one was synthesized with a cysteine residue at the N-terminus for coupling to rabbit albumin with MBS (Section 2.30). The positioning of the cysteine residue was due to the C-terminus predicted to be more immunogenic than the N-terminus with Predict 7 (Figure 5.2 B). Peptide FKLGSCYLYIINRNLKEI was coupled to rabbit albumin using glutaraldehye (Section 2.29). The peptides were synthesised with substitution of the internal cysteine residue with an alpha amino-butyric acid.
Peptide FKLGSCYLYIINRNLKEI corresponds to amino acid residues 567-584 of PY05757 (Figure 5.2 A, between solid black lines) and amino acid residues 2386-2403 of PFC0760c (Figure 5.2 A, between red dotted lines). The peptide was chosen for synthesis, as an alignment of the amino acid sequences of Plasmodium proteins having high sequence identity to Pca 96, revealed the sequence to be highly conserved across eight Plasmodium species (Chapter 3).
Peptide FKLGSCYLYIINRNLKEI did not appear to be a sequence that would be particularly immunogenic according to the predictions of Predict 7 (Figure 5.2 B), but it was decided to attempt to raise antibodies against it nevertheless, as it was thought useful to have a peptide that could potentially recognize the protein of interest in multiple Plasmodium species.
The peptide sequence was used to perform a BLAST search using the “search for short nearly exact matches” option at NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), to ensure that no sequence similarity existed between the peptide sequence and chicken proteins. Hypothetical proteins from P. falciparum, P. yoelii yoelii and P. chabaudi
chabaudi returned significant matches (Table 5.2), corresponding to proteins having high sequence similarity to Pca 96.
A
-4 -2 0 2 4
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
-0.2 -0.1 0 0.1 0.2
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
-4 -2 0 2 4
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
-0.2 -0.1 0 0.1 0.2
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
Amino acid residue number
-4 -2 0 2 4
1340 1345 1350 1355
Amino acid residue number Hydrophilicity and surface probability
-0.2 -0.1 0 0.1 0.2
Flexibility and antigenicity
Figure 5.1. Hydrophilicity, surface probability, flexibility and antigenicity predictions of the full length P. yoelii yoelii protein PY05757. Prediction of the hydrophilicity (), surface probability (▲), flexibility () and antigenicity (x) of protein PY05757 with Predict 7 (A). The position of the conserved sequence FKLGSCYLYIINRNLKEI is located between the solid black lines, and the position of peptide SDDDNRQIQDFE is located between the red dotted lines. An enlargement of the section where peptide SDDDNRQIQDFE is located is shown in (B).
B
-4 -2 0 2 4
2385 2390 2395 2400 2405
Amino acid residue number Hydrophilicity and surface probability
-0.2 -0.1 0 0.1 0.2
Flexibility and antigenicity
A
Amino acid residue number -4
-2 0 2 4
0 500 1000 1500 2000 2500 3000
-0.2 -0.1 0 0.1 0.2
0 500 1000 1500 2000 2500 3000
-4 -2 0 2 4
0 500 1000 1500 2000 2500 3000
-0.2 -0.1 0 0.1 0.2
0 500 1000 1500 2000 2500 3000
B
Figure 5.2. Hydrophilicity, surface probability, flexibility and antigenicity predictions of the full length P. falciparum protein PFC0760c. Prediction of the hydrophilicity (), surface probability (▲), flexibility () and antigenicity (x) of protein PFC0760c using Predict 7 (A). The position of the conserved sequence FKLGSCYLYIINRNLKEI is located between the dotted black lines. An enlargement the section where peptide FKLGSCYLYIINRNLKEI is located, is shown in (B).
Table 5.1. Significant matches returned by a BLAST search at NCBI* with the SDDDNRQIQDFE peptide
gi Number Name Strain E-value Identity
(%) gi|82596448|ref|XP_726266.1|
gi|23481602|gb|EAA17831.1|
Hypothetical protein PY05757
P. yoelii yoelii str. 17XNL
0.001 100
* BLAST search was performed using the „search for short nearly exact matches‟ option at NCBI.
Table 5.2. Significant matches returned by a BLAST search at NCBI* with the FKLGSCYLYIINRNLKEI peptide
gi Number Name Strain E-value Identity
(%) gi|3758855|emb|CAB11140.1|
gi|16805257|ref|NP_473285.1|
Hypothetical protein
P. falciparum 3D7
1x10-09 100
gi|82596448|ref|XP_726266.1|
gi|23481602|gb|EAA17831.1|
Hypothetical protein PY05757
P. yoelii yoelii 17XNL
1x10-09 100
gi|70941771|ref|XP_741132.1|
gi|56519318|emb|CAH81368.1|
Hypothetical protein
P. chabaudi chabaudi
1x10-09 100
gi|23508927|ref|NP_701595.1|
gi|23496764|gb|AAN36319.1|
Hypothetical protein PFL1165w
P. falciparum 3D7
0.64 61%
* BLAST search was performed using the „search for short nearly exact matches‟ option at NCBI.