Materials
Three groups of sera were selected at random from the institutional serum bank: (i) Sera with a positive sIgE test to HBV (i1 ≥0.1 kUa/L) and YJV (i3 ≥ 0.1 kUa/L) (n = 20); (ii) Sera with a positive sIgE test to YJV only (i3 ≥ 0.1 kUa/L) (n = 14); (iii) Sera with a positive sIgE test to HBV only (i1 ≥ 0.1 kUa/L) (n = 5). All patients had given their informed written consent to draw an additional serum sample.
Cloning of cDNA
Total RNA was isolated from yellow jacket (Vespula vulgaris) venom sacks using peqGold TriFast™ (Peqlab Biotechnologie, Erlangen, Germany). SuperScript III Reverse Transcriptase (Invitrogen, Karlsruhe, Germany) was used to synthesize cDNA. Full length Ves v 1 was amplified with Pfu DNA polymerase (Fermentas, St. Leon-Rot, Germany) using the primers 5'-GGACCCAAATGTCCTTTTAATTC-3' and 5'-AACCGCGGTTAAATTATCTTCCCCTTGTTA-3'. Full length Ves v 5 was amplified employing the primers 5'-AACAATTATTGTAAAATAAAATGTTTGAAA-3' and 5'-CTTTGTTTGATAAAGTTCCT-3'. An N-terminal 10-fold His-tag and a V5 epitope as well as 5' BamHI and 3' NotI restriction sites were added by PCR and the PCR product was subcloned into the pAcGP67-B baculovirus transfer vector (BD Pharmingen, Heidelberg, Germany) after restriction digest with BamHI and NotI.
Site directed mutagenesis
For generation of an inactive Ves v 1 form two amino acid residues in the potential active site were altered by using the QuikChange Site directed mutagenesis Kit (Stratagen, La Jolla, USA) according to the manufacturers' recommendations of the employing the primers 5'-CGATTAATTGGACATGGCTTAGGAGCACATG-3' and 5'-CATGTGCTCCTAAGCCATGTCCAATTAATCG-3' for S137G exchange and 5'-GAAATTATTGGGCTTGCTCCTGCTAGGCCTT-3' and 5'-AAGGCCTAGCAGGAGCAAGCCCAATAATTTC-3' for N165A exchange.
Recombinant baculovirus production and expression
Recombinant baculovirus was generated by cotransfection of Spodoptera frugiperda (Sf9) cells (Invitrogen) with BaculoGold bright DNA (BD Pharmingen) and the baculovirus transfer vector pAcGP67-B Ves v 1 or Ves v 5, respectively, according to recommendations of the manufacturer. High titer stocks were produced by three rounds of virus amplification and optimal multiplicity of infection (MOI) for recombinant protein expression was determined empirically by infection of Sf9 cells with serial dilutions of high titer virus stock.
Expression in baculovirus-infected Sf9 cells
High titer stocks of recombinant baculovirus containing the Ves v 1 or Ves v 5 coding DNA were used to infect Sf9 cells (1.5-2.0 × 106 cells per ml) in a 2000 ml suspension flask (400 ml suspension culture). For protein production the cells were incubated at 27°C and 110 rpm for 72 h.
Protein purification
Cellular supernatants were applied to a nickel-chelating affinity matrix (Ni-NTA-agarose, Qiagen, Hilden, Germany). After washing with NTA-binding buffer (50 mM sodium phosphate, pH 7.6, 500 mM NaCl) the protein was eluted with NTA-binding buffer containing 300 mM imidazole.
Enzymatic activity of rVes v 1
The enzymatic activity was assessed by use of the EnzChek Phospholipase A1 Assay Kit (Invitrogen) according to the recommendation of the manufacturer.
Biophysical analysis of rVes v 5
Dynamic light scattering (DLS) of rVes v 5 was performed using a Spectroscatterer 201 (RiNA GmbH, Berlin, Germany) equipped with a He-Ne laser providing radiation with a wavelength of 690 nm and an output power in the range of 10-50 mW. The sample (30 μl) with a protein concentration of 0.12 mg/ml in 50 mM sodium phosphate, 150 mM NaCl, pH 7.6 were placed in a quartz cuvette and measured at a constant temperature of 20°C.
Circular dichroism spectra were recorded at 20°C using a Jasco J-715 spectropolarimeter (Jasco, Groβ-Umstadt, Germany). A 1-mm optical pathlength quartz cell was used to obtain spectra in the far-UV region (190 to 260 nm) at a protein concentration of 0.015 mg/ml in 50 mM sodium phosphate, 150 mM NaCl, pH 7.65 μM. The CD spectra were acquired at a scan speed of 20 nm/min and a step resolution of 0.1 nm.
Immunoreactivity of human sera
For assessment of specific IgE immunoreactivity of human sera in ELISA, 384 well microtiter plates (Greiner, Frickenhausen, Germany) were coated with recombinant allergen, nApi m 1 (Latoxan, Valence, France) and the CCD marker MUXF-BSA (10 μg/ml) (provided by Siemens Healthcare Diagnostics, Los Angeles, USA) at 4°C overnight and blocked with 40 mg/ml skimmed milk powder in PBS at room temperature. Human sera were diluted 1:2 in PBS and applied for 4 hours at room temperature. Wells were rinsed 4 times with PBS and incubated with a monoclonal alkaline phosphatase-conjugated mouse anti-human IgE antibody (BD Pharmingen, clone G7-26) diluted 1:1000 in 20 mg/ml skimmed milk powder in PBS. Wells were again rinsed 4 times with PBS and substrate solution (5 mg/ml 4-nitrophenylphosphate, AppliChem, Darmstadt, Germany) was added. After 30 minutes plates were read at 405 nm.
Basophil activation test
The basophil activation test was essentially performed as recommended by the manufacturer (Bühlmann Laboratories, Basel, Switzerland). Stimulation with recombinant allergen was performed at protein concentrations of 0.1, 200 and 2000 ng/ml. YJV at a concentration of 50 ng/ml was used as positive stimulation control served (Bühlmann Laboratories) while plain stimulation buffer was used as negative stimulation control.
Other methods
SDS-PAGE and Western blotting as well as standard procedures in molecular biology were performed according to established protocols [11].