6.2 Results
6.2.8 Sequencing results of the scFv clones
The 30 clones selected for further analysis from the ELISA results were first assessed by colony and nested PCR and Alu1 restriction endonuclease digest before sequencing. Only 13 of the 30 scFv clones were sent for sequencing and were selected based on the following results. First colony PCR was performed on all 30 clones using the OP52 forward and M13 reverse primers to amplify the expected ~1000 bp scFv coding regions (van Wyngaardt et al., 2004). The resulting positive clones which gave ~1000 bp amplicons included five LDH clones (clone R4H13 was excluded); six GPADH clones, where clone R4H20 amplified an
~800 bp sequence; two anti-DEG (common PMT peptide) clones R3F12 and R3G7 and three clones against the PvPMT peptide (VYS peptide) R1F9, R1H1 and R1G12 (Figure 6.19).
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L R4H1 R4H8 R4H9 R1A9 R4H23 TG1 L R4H13 R4H20 R4H23 R1A11 R4H15 R4H18 TG1 L R1H1 R1G12 TG1
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A B C
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L 1 2 R4H1 R4H8 R4H13 R4H9 R1A9 R4H23 R4H13 R4H20 R4H23 R1A11 R4H15 R4H18 R3G6 R3F6 R3F12 R1B8
~1000 bp
~1000 bp
Anti-rPfLDH Anti-rPfGAPDH
Anti-CEV Anti-VYS
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L 1 2 R3G7 R3H11 R4D8 R4D2 R4C3 R4C7 R4B7 R4A7 R3F1 R1F9 R1H1 R1G12 R1H6 R4A4
Figure 6.19 Colony PCR of the selected clones against rPfLDH, rPfGAPDH and the PMT peptides Colony PCR was performed on the selected scFv clones against rPfLDH, rPfGAPDH and the PMT peptides (DEG, CEV and VYS) and assessed on a 1% agarose gel visualised with EtBr. An unrelated clone was picked and used as a positive control in lane 1, where lane 2 was an uninfected E. coli TG1 colony used as a negative control. The DNA ladder was loaded in the lane marked L. All other lanes were labelled with the respective clone abbreviation loaded in that lane.
Following successful colony PCR amplification of the scFv coding regions (~1000 bp amplicons), nested PCR was performed using a primer specific to the (GGGGS)3 linker coding sequence.
Figure 6.20 Nested PCR of the selected clones against rPfLDH, rPfGAPDH and the PvPMT peptide (VYS)
Nested PCR was performed on the selected scFv clones against rPfLDH (A), rPfGAPDH (B) and the PvPMT peptide (VYS) in (C) and assessed on a 3% agarose gel visualised with EtBr. An uninfected E. coli TG1 colony used as a negative control and the sample loaded in the TG1 lane. The DNA ladder was loaded in the lane marked L. All other lanes were labelled with the respective clone abbreviation loaded in that lane.
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L R4H1 R4H8 R4H9 R1A9 R4H23 R4H13 R4H20 R4H23 R1A11 R4H15 R4H18 R1H1 R1G12
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Successful amplification should result in amplicons around 500 bp in size as seen in Figure 6.20. This narrowed the number of clones to 13 of the original 30. Only the GAPDH clone R4H20 once again ran at ~800 bp for the colony PCR amplicon, but a nested PCR amplicon of approximately 500 bp was observed.To assess the identity of the 13 clones, the colony PCR amplicons were digested with the restriction endonuclease Alu1 (Finlay et al., 2006).
Depending on the number of Alu1 sites within each clone’s coding sequence, a unique
“fingerprint” or pattern may be observed when resolved on agarose as shown in Figure 6.21.
The resulting digest patterns suggested clones R4H1 and R4H8 to be similar; R4H9 and R4H23 and the two anti-PvPMT peptide clones R1H1 and R1G12. All 13 clones shown in Figure 6.21 were sent for sequencing using the colony PCR primer sets from Figure 6.19. The results for the LDH clones were presented first in Figure 6.22.
Figure 6.21 Alu1 digest of the selected clones against rPfLDH, rPfGAPDH and the PvPMT peptide (VYS)
Alu1 digest was performed on the colony PCR products of the selected scFv clones against rPfLDH, rPfGAPDH and the PvPMT peptide (VYS) and assessed on a 3% agarose gel visualised with EtBr. The DNA ladder was loaded in the lane marked L. All other lanes were labelled with the respective clone abbreviation loaded in that lane.
To link the sequencing results with the Alu1 digest results in Figure 6.21, the number of Alu1 restriction sites (AG^CT) within each of the anti-rPfLDH scFv coding regions was determined. The R4H1 clone coding sequence harboured two sites, which resulted in fragments of around 428, 368 and 233 bp which were visible in Figure 6.21. Clone R4H8’s coding sequence contained four sites, resulting in fragments of 428, 376, 259, 22 and 21 bp.
The three larger fragments were visible on the agarose gel, whilst the smaller fragments were too small to be observed. The R4H9 clone coding sequence contained two restriction sites resulting in three fragments of 404, 360 and 230 bp which were visible (Figure 6.21).
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CDR1 (VH) CDR2 (VH) CDR3 (VH)
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Germline R4H1 R4H8 R4H9 R1A9 R4H23 LA2 LD3 LB10 LE11 LC9
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Germline R4H1 R4H8 R4H9 R1A9 R4H23
Germline R4H1 R4H8 R4H9 R1A9 R4H23
Germline R4H1 R4H8 R4H9 R1A9 R4H23
Germline R4H1 R4H8 R4H9 R1A9 R4H23
Germline R4H1 R4H8 R4H9 R1A9 R4H23
Figure 6.22 Alignment of the anti-rPfLDH scFv clones’ translated sequences with a chicken germline immunoglobulin and the Chiliza et al. (2008) anti-LDH scFv sequences The translated amino acid sequence for the anti-rPfLDH clones were aligned with a chicken IgG germline amino acid sequence in (A). The linker region (G4S)3 and the framework regions 1 to 4 were underlined, with VH and VL for heavy and light chains respectively. The complementarity determining regions (CDR) 1 to 3 for both the VH and VL chains were box- shaded and in bold. Each of the CDRs was additionally aligned with anti-LDH scFv CDRs isolated from an immune library by Chiliza et al. (2008) in (B). In the alignments “.” represent identical residues to the germline sequence, letters represent an amino acid substitution, “-”
represent gaps in the alignment and “X” represent an undetermined amino acid codon from the sequencing results.
164 The R1A9 clone had two restriction sites within its coding sequence and resulted in three fragments of around 359, 261 and 257 bp respectively. The only aberration in the Alu1 results was the larger band within the R1A9 sample, which may have been as a result of incomplete digestion that may result in a band of approximately 518 bp (261+257 bp). The size difference between the 261 and 257 bp fragments is unlikely to be visible and the band at around 260 bp on the gel most likely includes both fragments. Finally the R4H23 clone against rPfLDH had two restriction sites within its coding sequence, resulting in three bands of around 402, 358 and 234 bp respectively, which also correlated with the results of the restriction digest shown in Figure 6.21.
The heavy and light chains in scFv coding sequences each code for as set of four frame work regions (FR1 to 4) and three complementarity determining regions (CDR1 to 3) as indicated in Figure 6.22 (A). Most conservation between sequences is expected for the frame work regions, which held true for most clones except the R4H23 clone which contained unique FR1 to three regions for its heavy chain. Most amino acid variations amongst all five clones were within the CDRs and in particular the CDR2 and 3 of both the light and heavy chains.
The least variation was within the CDR1 of the light chain in all cases. Interestingly only clones R4H1 and R4H8 had identical CDRs and only varied within their heavy chain CDR1 and 2 respectively. None of the clones resembled those isolated from an immune chicken library screened with rPfLDH by Chiliza et al. (2008) as shown in the alignment in Figure 6.22 (B) though there were a few conserved amino acids within the respective CDRs.
Analysis of the anti-rPfGAPDH scFv clones was done next. Based on the sequencing results, clone R4H20 had three Alu1 restriction sites which should have resulted in four fragments of 434, 172, 153 and 23 bp respectively. From Figure 6.21 only two of these bands were visible, namely the 434 bp and 172 bp fragments. The R1A11 clone contained two restriction sites resulting in three fragments of 428, 398 and 240 bp respectively, which were all visible on the agarose gel. The individual sequences are shown in Figure 6.23. The R4H18 clone was only successfully sequenced in the reverse direction, hence an incomplete sequence was shown in Figure 6.23. A high degree of similarity between it and the R1A11 clone was observed, however, and the CDR1 to 3 of the heavy chain were almost identical to those of the R1A11 clone. What was more interesting was that clone R1A11 and clone R4H1 against rPfLDH (Figure 6.22) shared 85% identity, with identical CDRs throughout.
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VHFR1
VHFR3
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CDR3 Germline
R4H20 R1A11 R4H18
Germline R4H20 R1A11 R4H18
Germline R4H20 R1A11 R4H18
Germline R4H20 R1A11 R4H18
Germline R4H20 R1A11 R4H18
Figure 6.23 Alignment of the anti-rPfGAPDH scFv clones’ translated sequences with a chicken germline immunoglobulin sequence
The translated amino acid sequence for the anti-rPfGAPDH clones were aligned with a chicken IgG germline amino acid sequence. The linker region (G4S)3 and the framework regions 1 to 4 were underlined, with VH and VL for heavy and light chains respectively. The complementarity determining regions (CDR) 1 to 3 for both the VH and VL chains were box-shaded and in bold. In the alignments “.” represent identical residues to the germline sequence, letters represent an amino acid substitution, “-” represent gaps in the alignment and “X”
represent an undetermined amino acid codon from the sequencing results.
Finally the anti-PvPMT peptide scFv clones were assessed as shown in Figure 6.24. Both clones harboured only single Alu1 restriction sites. This was predicted to result in 648 and 393 bp fragments for clone R1H1 and 644 and 397 bp fragments for clone R1G12 which correlated with the bands detected on the agarose gel in Figure 6.21.
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VHFR1
VHFR3
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VHFR4 Linker VLFR1
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R1H1 R1G12
Germline R1H1 R1G12
Germline R1H1 R1G12
Germline R1H1 R1G12
Germline R1H1 R1G12
CDR1
CDR2 CDR3
CDR3 CDR1
CDR1 CDR2
CDR3
Figure 6.24 Alignment of the anti-PvPMT peptide (VYS) scFv clones’ translated sequences with a chicken germline immunoglobulin sequence
The translated amino acid sequence for the anti-PvPMT peptide (VYS) scFv clones were aligned with a chicken IgG germline amino acid sequence. The linker region (G4S)3 and the framework regions 1 to 4 were underlined, with VH and VL for heavy and light chains respectively. The complementarity determining regions (CDR) 1 to 3 for both the VH and VL chains were box-shaded and in bold. In the alignments “.” represent identical residues to the germline sequence, letters represent an amino acid substitution, “-” represent gaps in the alignment and “X”
represent an undetermined amino acid codon from the sequencing results.
The two clones share an identical CDR2 region in their light chains, with several amino acid differences in their remaining CDRs. Importantly these were the only two scFv sequences which were in frame for both the vector encoded N-terminal (LLLLAAQPA) and C-terminal cmyc sequence (EQKLISEEDLN).
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