Chapter IV Discussion and Conclusion
4.2 Conclusion
In brief, we successfully linked the Der p 1 and Der p 2 genes with an α-helix
forming nucleotide sequences. Via constructed in a pPICZαA vector, it was
electroporated into P. pastoris wild strain. A high productivity strain (strain No. 33) for
production of fusion allergen was chosen. The productivity of the fusion allergen in
Hinton’s flask was 35.5 μg/mL after 72 h of methanol induction. A high cell density
culture in the Bioflo110 fermentor was achieved which caused to 203 mg/L fusion
allergen productivity at 60 h and got a 427 g/L wet biomass at 132 h. This combination
of the major allergens in Dermatophagoides pteronyssinus is suspected to be
developed as an efficient vaccine for allergen disease treatment.
47
Tables and Figures
48
Table 3House dust mite allergens. (Thomas et al., 2002)
Group Biochemical function MW cDNA1
(SDS-PAGE) Species2 IgE binding3
8 Glutathione-S-transferase 26,000 Dp 40
9 Collagenolytic serine protease no cDNA, (30 000) Dp 90
10 Tropomyosin 37,000 Dp, Df 50–95
11 Paramyosin 96,000 (92,000,
98,000) Df, Bt 80
12 Unknown 14,000 Bt 50
13 Fatty acid-binding protein 15,000 Bt, Ld, As 10–23 14 Vitellogenin/apolipophorin-like 177,000 (variable) Df, Dp, Em 90 15 98,000 Chitinase 62,500 (98,000,
105,000) Df 70
16 Gelsolin 55 Df 35
17 Ca-binding EF protein 30 Df 35
18 Chitinase 60,000 Df 60
19 Anti-microbial peptide 7,000 Bt 10
1 MW calculated from cDNA (SDS-PAGE of natural allergen, if different).
2 Allergen described for the species designated by initials: Dermatophagoides pteronyssinus, Dermatophagoides farinae, Euroglyphus maynei,Dermatophagoides siboney, Dermatophagoides microceaus, Lepidoglyphus destructor, Blomia tropicalis, Tyrophagus putrescentiae, Glycophagusdomesticus, Ascaris siro.
3 Binding frequency (% patients, variation due to patient selection).
49
Table 2 Recombinant allergens expressed in various systems. (Schmidt and Hoffman, 2002)
Expressed in Allergen Comments
E. coli Api m 1 Renatured to full enzyme and IgE-binding activities Api m 2 Only 20–30% enzyme activity after renaturation Cyp c 1 Produced in fully active form when calcium added Ara h 1 Codon usage requires specially engineered strain Bet v 1 Isoforms with varying IgE-binding activity Cor a 1 Bet v 1 cross-reactive, isoforms
Alt a 1 IgE-reactive, glycosylated Mus m 1 Native conformation Bla g 4 High yield
Fel d 1 Glycosylated
Der p 1 Hyperglycosylated precursor Ves v 5 High yield, native conformation Saccharomyces Der p 1 Insoluble
Der p 2 IgE-binding activity
Procalin Immunoreactive
Baculovirus Api m 2 Full enzymatic and IgE-binding activities
Sol i 2 IgE-reactive, native conformation, natural cleavage of initiation sequence
Fel d 1 Fully immunoreactive Lep d 2 Isoforms produced
Mal f 1 Similar to that expressed in E. coli
50 Table 3 Primer design and the linker sequence.
Fragment nucleotides sequence
pEcoRI-Der p 1 5'-AAG-GAATTC-ACT AAC GCC TGC AGT ATC AA-3' pDer p 1-XhoI 5'-CTG-CTCGAG-GAG AAT GAC AAC ATA TGG AT-3'
pXhoI-linker 5'-ACA-CTCGAG-GGT TCT ACC TCT-3 plinker-SacII 5'-TTA-CCGCGG-GAT ACC AGA ACC-3'
pSacII-Der p 2 5'-AAT-CCGCGG-GAT CAA GTC GAT GTC AAA GA-3' pDer p 2-XbaI 5'-ACA-TCTAGA-CC-ATC GCG GAT TTT AGC ATG AGT-3' Linker sequence 5'-ACA-CTCGAG-GGT TCT ACC TCT GGT GGT TCT ACC TCT GGT
GGT TCT ACC TCT GGT TCT GGT TCT GGT ATC-CCGCGG-TAA-3'
51
Table 4 Comparisons of rDer p 1 and rDer p 2 expressed in different hosts
allergen system concentration year
Der p 1 E. coli low 1988
Der p 1 Drosophila cells 20 μg/mL 2000
Der p 1 mammalian cells 34 μg/mL 2001
Der p 1 Pichia pastoris 70 μg/mL 2002
Der p 1 rice 50μg/grains 2008
Der p 2 E. coli 50 μg/mL 1997
Der p 2 Saccharomyces cerevisiae 7 μg/mL 1998
Der p 2 Tobacco 8 μg/mL 2002
Der p 1+Der p 2 Pichia pastoris up to 203 μg/mL 2008
52
Figure 1 pPICZαA vector. (A) is the multiple cloning site of pPICZαA and (B) is the vector map. The figure and the complete sequence of pPICZαA is available for downloading from Invitrogen company’s World Wide Web site (www.invitrogen.com).
Figure 2 Schematic representation of Der p 1 and 2 fusion allergen.
53 Figure 3 Sequencing result of fusion gene.
54
Figure 4 Results of the Zeocin selection. Thirty five strains cultured on YPDZ plate for 4 days and the Zeocin concentrate was from 50 μg/mL to 1000 μg/mL.
55
Figure 5 Colony PCR result. 5-1, AOX primer. 5-2, Der p 1+linker primer. 5-3, linker+Der p 2 primer
5-1
5-2
5-3
56 Figure 6 Standard curve of Der p 2 ELISA.
57
Figure 7 Selection results of 9 strains cultured in a small scale methanol induction.
58
Figure 8 Fusion allergen expression efficiency of strain 6, 10 and 33.
59
Figure 9 Western blotting results of strain 33 cultured in Hinton’s flasks.
60 Figure 10 Strain 33 cultured in Hinton’s flasks.
.
61 Figure 11 Strain 33 cultured in a fermentor.
62
Figure 12 ELISA and the Western results of strain 33 cultured in a fermentor.
63
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Appendix
77
Media for yeast:
Yeast Extract Peptone Dextrose broth (YPD, per liter)
Yeast Extract 10 g
Peptone 20 g
Glucose 20 g
Yeast Extract Peptone Dextrose agar (YPD, per liter)
Yeast Extract 10 g
Peptone 20 g
Glucose 20 g
Agar 20 g
Yeast Extract Peptone Dextrose agar with Zeocin (YPDZ, per liter)
Yeast Extract 10 g
Peptone 20 g
Glucose 20 g
Agar 20 g
*autoclaved and cool solution to below 60oC before antibiotics were added
Zeocin (100 mg/ mL stock) 1 mL
Increase the usage of Zeocin stock to formulate different concentration of Zeocin YPD agar with sorbitol and Zeocin (YPDSZ, per liter)
Yeast Extract 10 g
Peptone 20 g
Sorbitol 182.2 g
Agar 20 g
* add water to 900 mL autoclaved and cool solution to below 60oC
Glucose (20%) 100 mL
Zeocin (100 mg/ mL stock) 1 mL
78
BMG broth (per liter)
Potassium phosphate 100 mM
* adjust pH to 6.0
Yeast Nitrogen Base 13.4 g
d-biotin 0.4 mg
glycerol 10 mL
* the above 3 were filter steriled by 0.22 mm filter membrane
BMM broth (per liter)
Potassium phosphate 100 mM
* adjust pH to 6.0
Yeast Nitrogen Base 13.4 g
d-biotin 0.4 mg
methanol 5 mL
* the above 3 were filter steriled by 0.22 mm filter membrane
Media for E. coli
Luria-Bertani agar (LB, per liter)
Tryptone 10 g
Sodium Chloride 10 g
Yeast Extract 5 g
* adjust pH to 7.5
Agar 15 g
LB broth with ampicillin (LBA, per liter)
Tryptone 10 g
Sodium Chloride 10 g
Yeast Extract 5 g
* adjust pH to 7.5, autoclaved and cool solution to below 60oC before antibiotics were added
Ampicillin (50 mg/mL stock) 2 mL
79
LB agar with ampicillin (LBA, per liter)
Tryptone 10 g
Sodium Chloride 10 g
Yeast Extract 5 g
* adjust pH to 7.5
Agar 15 g
*autoclaved and cool solution to below 60oC before antibiotics were added
Ampicillin (50 mg/mL stock) 2 mL
Low salt LB Agar with Zeocin (LSLBZ, per liter)
Tryptone 10 g
Sodium Chloride 5 g
Yeast Extract 5 g
* adjust pH to 7.5
Agar 15 g
*autoclaved and cool solution to below 60oC before antibiotics were added
Zeocin (100 mg/mL stock) 250 μL
* 1X TAE buffer is obtained by diluting in ddH2O 50 times
A solution (per 50 mL)
Acrylamile 14.6 g
Bis 0.4 g
80
Bromophenol blue 1 mg
1X TBE 5 mL
Glycerol 5 mL
Plasmid construction
PCR
Super-Therm DNA Polymerase (Bertec, Taipei, Taiwan)
81 dNTP mixture (Viogene, CA, USA)
10x PCR reaction buffer (Bertec, Taipei, Taiwan)
Restriction enzyme
yT&A cloning vector kit (Yeastern Biotech, Taipei, Taiwan) T4 DNA ligase (Takara, Shiga, Japan)
QIAprep® Spin Miniprep Kit (Qiagen, Hilden, Germany) Gel extraction kit (Geneaid, Taipei, Taiwan)
T4 DNA ligase (Takara, Shiga, Japan)
Protein purification and detection
Protein quantity
Bradford (Bio-Rad, CA, USA)
Western blotting
Mouse anti-Der p 2 antibody generated hybridorma (kindly provided by Professor Chiang)
Or Rabbit anti-Der p 2 antibody made by Genesis Biotech Inc.
Western lightning-Chemical Reagent Plus (PerkinElmer Life Science, Inc., MA, USA) Hybond-C Extra membrane (Amersham, London, UK)
Goat anti-Rabbit IgG (H+L) HRP conjugated (Chemicon, CA, USA) Transfer buffer: 192 mM Glycine, 25mM Tris, 20% Methanol 10x TBS: 0.5M Tris Base, 9% NaCl, pH 7.6
Blocking buffer: 1X TBS, 5% skim milk Wash buffer (TBST): 1X TBS, 0.25 % Tween20
ELISA
Mouse anti-Der p 2 antibody
Rabbit anti-Der p 2 antibody made by Genesis Biotech Inc.