Trifluoroacetic acid (TFA), α-cyano-4-hydroxycinnamic acid (CCA), acetonitrile (ACN) and dithiothreitol (DTT) were obtained from Sigma (St. Louis, MO, USA). Immobiline drystrips, ammonium persulfate, urea, agarose, glycerol, bromophenol blue, iodoacetamide (IAA), silver nitrate, 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane (CHAPS), and N, N, N’, N’-tetramethylethylenediamine (TEMED) were from Amersham Pharmacia Biotech (Uppsala, Sweden). Acrylamide, Bis, Tris, glycine, SDS and SDS-PAGE protein standards were from Bio-Rad (Hercules, CA).
Egg extract preparation
Egg extract was prepared from the eggs of about 1–2 weeks before hatching of newborns. After being washed with an insect physiological buffer (in g/L: NaCl 8.19, KCl 0.37, CaCl2 0.56, MgCl2·6H2O 0.2, Glucose 0.9, HEPES 2.4, pH7.25) twice, the eggs were homogenized in a neutral buffer of weak ionic strength (10 mM PBS buffer) or ddH2O with a mortar and pestle. The homogenate was centrifuged at 10 000 g for 10 min at 4°C and the pellet was repeatedly homogenized and extracted twice. The supernatants were pooled and lyophilized.
Determination of protein content and hydrolase activity
Protein content of the extract was quantitatively determined using Bradford method . The determinations of protease , alkaline phosphatase , acid phosphatase , acetylcholine esterase  and hyalurinidase  were performed, respectively, according to the methods described previously in literature.
Biological assay of egg extract
The extract sample was intra-abdominally injected into mice (2–6 mg/kg body weight) and cockroaches (10 μg/g body weight) to determine whether the egg extract contained components toxic to animals. Determination of LD50 in mice was conducted according to the methods described by Schweitz  and Liang et al. . Briefly, the accurately weighed extract powder was dissolved in physiological saline and centrifuged at 10 000 × g for 10 min. The supernatant was used for the experiment. For LD50 determination, 48 mice (albino Kunming) of both sexes, weighing 20 ± 2 g, were randomly divided into eight groups, each of which consisted of six mice. Seven groups were used as experimental groups to which the extract sample solution was administrated intraperitoneally as single doses of 2.211, 2.601, 3.060, 3.600, 4.235, 4.983 and 5.862 mg/kg body weight, respectively. The eighth group was used as control and injected with physiological saline. Lethality in mice was observed during a 24 h period after injection. The LD50 value was determined based on the lethality in six animals at each dose level.
Furthermore, the neurotoxicity of the extract was analyzed using mouse isolated phrenic nerve-hemidiaphragm preparations according to the methods described previously . Briefly, adult Kunming albino mice were killed by cervical dislocation. The phrenic nerve-hemidiaphragm preparation was isolated and placed in a small plexiglas chamber immersed in Tyrode’s solution with or without adding extract sample, continuously bubbled with a mixture of 95%O2 and 5%CO2, and maintained at 30-32°C. Electrical stimulation was applied to the phrenic nerve with a suction electrode (supramaximal voltage, 2 ms duration, square wave). The resulting twitch responses of the phrenic muscle were transformed into an electric signal by a mechanical-electric transducer. Signals were amplified and recorded with a signal process system (BL-420 S, China).
Whole-cell patch-clamp assays
Whole-cell patch-clamp technique was employed to detect the effects of the egg extract on the ion channels in rat dorsal root ganglion (DRG) neurons. The DRGs were acutely isolated from 30-day-old Sprague–Dawley rats of either gender and the neurons prepared from the DRGs were maintained in short-term primary culture according to the methods described previously [38, 39]. The patch-clamp pipettes with a tip resistance of 2.0-3.0 MΩ were made of borosilicate glass capillary tubes. The extract-containing solutions of 10 μL volume were applied by pressure injection with a microinjector (IM-5B, Narishige, Tokyo, Jianpan) through a micropipette (20 μm in tip diameter) placed about 100 μm away from the cells under study . Ion channel currents were recorded at room temperature (20–25°C). All data were presented as means ± SD, and n was used to represent the number of independent experiments and was generally ≥5.
In order to detect the effects of egg extract on sodium currents in DRG cells, the inward sodium currents were elicited by a 50 ms step depolarization to −10 mV from a holding potential of −80 mV every 5 seconds. When the influences of the extract on the current–voltage (I-V) relationship were investigated, the sodium currents were induced by 50 ms depolarization steps to various potentials from a holding potential of −80 mV. Test potentials ranged from −80 to +70 mV in 10 mV increments. The patch-clamp pipette was filled with a solution (pH 7.2) containing (in mM) CsCl 145, MgCl2·6H2O 4, HEPES 10, EGTA 10, glucose 10, ATP 2, and the bath solution (pH 7.4) contained (in mM) NaCl 145, KCl 2.5, CaCl2 1.5, MgCl2·6H2O 1.2, HEPES 10, EGTA 10, glucose 10. In view of the fact that the larger DRG neurons tend to express tetrodotoxin-sensitive (TTX-S) sodium channels whereas the smaller ones tend to express tetrodotoxin-resistent (TTX-R) sodium channels , the DRG neurons with diameter greater than 40 μm or smaller than 20 μm were used to detect the effects of the egg extract on TTX-S sodium currents and TTX-R sodium channels, respectively. Tetrodotoxin (0.2 μM) was added to the bath solution to separate TTX-R sodium currents from TTX-S sodium currents .
For investigating the effects of the egg extract on potassium currents, the potassium currents in DRG cells were elicited by a 500 ms depolarization to +30 mV from a holding potential of −80 mV every 5 seconds. When the effects of the extract on the current–voltage (I-V) relationship were investigated, the potassium currents were induced by 50 ms depolarization steps to various potentials from a holding potential of −80 mV. Test potentials ranged from −80 to +70 mV in 10 mV increments. The suction pipette solution contained (in mM) KCl 135, KF 25, NaCl 9, MgCl2 1, EGTA 1, HEPES 10 and ATP-Na2 3, adjusted to pH 7.4 with 1 M KOH, and the external bath solution contained (in mM) NaCl 150, KCl 30, CaCl2 5, MgCl2 4, TTX 0.3, HEPES 10 and D-glucose 10, adjusted to pH 7.4 with 1 M NaOH.
For recording calcium currents in DRG neurons in the presence and absence of the egg extract, the total calcium currents in rat DRG cells were elicited by a 150 ms depolarization to 0 mV from a holding potential of −90 mV. Low-voltage-activated (LVA) calcium channels were activated by a 100 ms step depolarization to −50 mV from a holding potential of −90 mV, whereas high-voltage-activated (HVA) calcium channels were activated by depolarization from a holding potential of −40 mV to 0 mV. The pipette internal solution contained (in mM) Cs-methane sulfonate 110, phosphocreatine 14, HEPES 10, EGTA 10, ATP-Mg 5, adjusted to pH 7.3 with CsOH, and the external bath solution contained (in mM) BaCl2 10, tetraethylammonium (TEA)-Cl 125, TTX 0.3 and HEPES 10, adjusted to pH 7.4 with TEA-OH.
MALDI TOF MS analysis
MALDI TOF mass spectrometry was used to detect the proteins and peptides with molecular masses below 10 kDa. The low-molecular-mass fraction was prepared by ultrafiltrating the egg extract with a centrifugal filter (10 000 MWCO, Millipore). Mass spectrometric analysis was performed on an ultraflex TOF/TOF mass spectrometer (Bruker Daltonics Inc.). Acquisition operation mode was linear. Sample solution was mixed with the saturated α-cyano-4-hydroxycinnamic acid solution (prepared with 50% ACN containing 0.1% TFA) at a ratio of 1:1 and 1 μL of the mixture solution (about 1 μg proteins/peptides) was applied onto the sample carrier for the analysis.
SDS-PAGE of extract and venom
SDS-PAGE of the egg extract and venom was performed according to the method of Laemmli  under denaturing conditions on a 4.8% stacking gel and an 11.5% separation gel. Aliquots of lyophilized extract and venom (each containing 100 μg proteins) were separately dissolved in 30 μL of sample buffer (50 mM Tris–HCl, pH 6.8, 65 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2%SDS, and a trace of bromophenol blue) and boiled for 3 min. The sample solutions were centrifuged at 10 000 × g for 15 min and the supernatants were loaded into the parallel gel wells. The SDS-PAGE was run at 25 mA on the stacking gel and at 45 mA on the separating gel. After carrying out the electrophoresis, the separated proteins were visualized by Coomassie brilliant blue G-250 staining. A prestained protein ladder (from Bio-Rad) was used as standard molecular mass markers.
2D-PAGE of extract
The lyophilized egg extract was separated with two-dimensional gel electrophoresis (2D-PAGE) according to the method previously described  to gain more detail information on the large protein components in the eggs. 500 μg of the extract powder was dissolved in about 350 μL of rehydration solution (8 M urea, 4% (w/v) CHAPS, 65 mM DTT, 0.5% (v/v) IPG buffer, 0.5% pharmalyte, a trace of Bromophenol Blue). The mixture solution was clarified by centrifugation at 10 000 × g for 10 min. Commercial 18 cm IPG strip (Bio-Rad) with a linear range of pH3-10 was rehydrated overnight with the sample solution. Isoelectric focusing was performed in a Bio-Rad Protean isoelectric focusing unit according to the method described by the manufacturer. The conditions for isoelectric focusing were as follows: 30 V for 14 h; 500 V for 1 h; 1000 V for 1 h; 8000 V for up to 32000 Vh. Before running the second dimension, the IPG strip was placed in a tray and the egg proteins in the strips were reduced and alkylated by sequential incubation in equilibration solution A (0.05 M Tris–HCl, pH6.8, 8 M urea, 30% glycerol, 1% SDS and 0.2% DTT) for 15 min, and in equilibration solution B (0.05 M Tris–HCl, pH6.8, 8 M urea, 30% glycerol, 1% SDS, 3% IAA and a trace of bromophenol blue) for another 15 min. For the second electrophoresis separation, the strip was embedded on top of the 2D gel and covered with agarose. Second–dimensional SDS-PAGE was performed on 5% polyacrylamide stacking gel (25 mA per gel) and 12% polyacrylamide separating gel (50 mA per gel) until the dye front reached near the bottom of the gel at a temperature of 10°C. The separated egg proteins in the gel were visualized by Coomassie brilliant blue G-250 staining.
All procedures conformed to the Guidelines of the National Institutes of Health Guide for the Care and Use Laboratory Animals. The present study was approved by the Ethics Committee on the Use and Care of Animals of the Hunan Province, P. R. China.