Abstract -The aim of this study was to establish and optimize the two-dimensional electrophoresis (2-DE) technology for hydatid fluid proteome of the Echinococcus granulosus, and obtain 2-DE map of hydatid fluid proteome. Total proteins of hydatid fluid were extracted by lyophilization. A series of important factors, such as sample preparation, protein quantities, pH range of immobilized pH gradient (IPG) strip and extraction methods, were optimized to improve the resolution and repeatability. Two-dimensional electrophoresis maps were analyzed after staining. The 2-DE profiles with high resolution and good repeatability were obtained, when the hydatid fluid dealt with ReadyPrep TM 2-D Cleanup Kit were analyzed with the established 2-DE using 400µg of quantitative loading and IPG strips pH7-10. We identified 30 protein spots using PDQuest 8.0 2D analysis software. The molecular weight of most of these proteins ranged from 43 to 97kDa and the isoelectric points of these proteins ranged from 5 to 9. An optimized 2-DE system is set up successfully in this study, electrophoresis pattern of which shows a high resolution and good repeatability, and can be used for the study of E. granulosus proteomics effectively

Jinhu Wu

Juyi Li

Ling Chen

Xiufang Wang

Yi Wang

Zhaoqing Zhang

CiteSeerX

Pakistan J. Zool., vol. 46(5), pp. 1249-1254, 2014 
 
Establishment and Optimization of Two-dimensional Electrophoresis 
Technique in Hydatid Fluid Proteome of Echinococcus granulosus 
 
Juyi Li1, Xiufang Wang2, Yi Wang1*, Zhaoqing Zhang2, Ling Chen3 and Jinhu Wu3 
1Department of Pharmacy, The Central Hospital of Wuhan, Wuhan, Hubei Province, China. 
2Department of Rehabilitation, The Third Hospital of Wuhan, Wuhan, Hubei Province, China. 
3Department of Pharmacy, The Third Hospital of Wuhan, Wuhan, Hubei Province, China. 
 
 Abstract - The aim of this study was to establish and optimize the two-dimensional electrophoresis (2-DE) 
technology for hydatid fluid proteome of the Echinococcus granulosus, and obtain 2-DE map of hydatid fluid 
proteome. Total proteins of hydatid fluid were extracted by lyophilization. A series of important factors, such as 
sample preparation, protein quantities, pH range of immobilized pH gradient (IPG) strip and extraction methods, were 
optimized to improve the resolution and repeatability. Two-dimensional electrophoresis maps were analyzed after 
staining. The 2-DE profiles with high resolution and good repeatability were obtained, when the hydatid fluid dealt 
with ReadyPrepTM 2-D Cleanup Kit were analyzed with the established 2-DE using 400µg of quantitative loading and 
IPG strips pH7-10. We identified 30 protein spots using PDQuest 8.0 2D analysis software. The molecular weight of 
most of these proteins ranged from 43 to 97kDa and the isoelectric points of these proteins ranged from 5 to 9. An 
optimized 2-DE system is set up successfully in this study, electrophoresis pattern of which shows a high resolution 
and good repeatability, and can be used for the study of E. granulosus proteomics effectively.  
 
Key words: Proteomics, cestode, immobilized pH gradient, cystic echinococcosis 
 
 
INTRODUCTION 
 
 Cystic echinococcosis (CE) is a zoonosis 
caused by the larval form of the cestode 
Echinococcus granulosus (Li and Zhao, 2012). The 
domestic life cycle of E. granulosus is maintained 
through definitive host and intermediate hosts. The 
adult form lives in the small intestine of a carnivore 
(definitive host). A wide range of mammal species 
(intermediate host), including humans, may acquire 
the infection through accidental ingestion of eggs 
containing infective oncospheres of E. granulosus. 
The egg hatches in the intestinal tract and the 
oncosphere is freed, migrates through the intestinal 
wall, and gains access via the circulatory system to 
the muscles, brain and other tissues of the host, 
where it transforms into the metacestode form or 
cysticercus. The cyst of cysticercus forms a 
relatively stable internal environment to avoid 
damage to the larvae from the host immune system 
 
_______________________________ 
* Corresponding authors: Yi Wang 694531611@qq.com  
 
0030-9923/2014/0005-1249 $ 8.00/0 
Copyright 2014 Zoological Society of Pakistan 
 
(Li et al., 2013). The disease has a worldwide 
distribution, with a considerable impact on both 
human and animal health and causes important 
socio-economic consequences in endemic areas (Shi 
et al., 2009). 
 Hydatid-cyst fluid (HCF) is a clear or clear 
yellow liquid with antigenic properties and an 
important component of the internal environment 
and fills the entire cyst. HCF provides needed 
nutrition for larval growth, playing an important role 
in their life cycle of E. granulosus. Previous studies 
focused on livestock such as sheep and cattle. 
Shepherd and McManus (1987) discovered five 
major subunit antigens of relative molecular mass 
(Mr) 12,000, 16,000, two at 20,000, and 38,000 in 
HCF through sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) 
from E. granulosus. Zhu et al. (1989)  used 
improved two-dimensional polyacrylamide 
electrophoresis to identify 111 proteins in the liver 
HCF of infected sheep , and 130 HCF proteins were 
identified from fertile cysts of sheep and human 
origin and infertile cysts from cattle (Aziz et al., 
2011). Chemale et al. (2003) attempted to analyze 
proteins in liver HCF of cattle but failed to establish 
J. LI ET AL.   1250
a 2-DE database because of the effect of highly 
abundant albumin and immunoglobulin.  
 HCF is a complex biological mixture 
containing a wide range of proteins of both parasite 
and host origin, playing an important role in the life 
cycle of E. granulosus, however, the research on 
HCF proteins is limited, because of the effect of 
highly abundant albumin and immunoglobulin 
(Chemale et al., 2003). Therefore, it is very 
necessary to find effective methods to dissociate and 
identify the proteins of HCF, and it may help reveal 
new candidates for immunodiagnosis and vaccines. 
 Proteomics offers a set of tools for 
investigating proteins from HCF. Thus, a series of 
important factors, such as sample preparation, 
protein quantities, pH range of IPG strip and 
extraction methods, were optimized to improve the 
resolution and repeatability, to establish and 
optimize the 2-DE technology for hydatid fluid 
proteome of the E. granulosus, and obtain satisfied 
2-DE map of hydatid fluid proteome, which stands 
as a valuable resource for further hydatid fluid 
proteomics research of E. granulosus. 
 
MATERIALS AND METHODS 
 
Purification of hydatid-cyst fluid (Li et al., 2013) 
 Human HCF was collected after the surgical 
removal of fertile cysts from patients with cystic 
hydatid disease in The General Hospital of Ningxia 
Medical University. In total, 21 cysts of different 
sizes were isolated in a germ-free environment, and 
55ml of cyst fluid was aspirated from these cysts 
using sterile needles under aseptic conditions and 
centrifuged at 10,000g for 15min at 4°C to remove 
particles. The supernatant fluid was stored at -80°C 
until use. This study had ethical approval and all 
subjects gave informed consent to participate.  
 
2D Sample preparation 
 First, 20 mL of cyst liquid was pre-frozen at -
85°C for 5 h and then place in the freeze-dryer (-
60°C vacuum) overnight. After 24h, the cyst liquid 
was freeze-dried into a powder. The powder was 
lysed using lysis buffer [9mol/L urea, 4% 3-(3-
cholamidopropyl) dimethyl-ammonio-1-propane 
sulfonate (CHAPS), 1% dithiothreitol (DTT), and 
0.5% protease-inhibitor cocktail], fully oscillated 
and blended at 4°C for 1h, and centrifuged at 
12,000g at 4°C for 30min. The final supernatant 
fluid was stored at -85°C until use. TCA-Acetone 
Extraction: the above supernatant was added 
directly to an equal volume of 20% TCA in acetone. 
Proteins were precipitated overnight for at least at -
20°C. Precipitated protein was centrifuged at 
12,000g at 4°C for 30min, washed three times in 
1mL in ice-cold acetone, centrifuged at 12,000g at 
4°C for 15min, the precipitation were re-solubilized 
in immobilized pH gradient (IPG) rehydration buffer. 
 To remove salt, lipids, and undesired 
detergents, cleanup was performed with the 
ReadyPrepTM 2-D Cleanup Kit (BIO-RAD). 
 The AurumTM Serum Protein Mini Kit (BIO-
RAD) was used to remove the most abundant 
proteins.  
 The protein pellets were re-solubilized in IPG 
rehydration buffer (7M urea, 2M thiourea, 2% 
CHAPS, 50mM DTT, 0.2% Bio-Lyte ampholyte, 
0.001% bromophenol blue) and then centrifuged. 
The supernatant was held at 4°C. The total protein 
concentration for each sample was determined using 
the Bradford assay. Bovine serum albumin was used 
at 9 different concentrations (0.25, 0.5, 1, 1.5, 2, 3, 4, 
5 and 6 mg/mL) to prepare a protein standard. After 
diluting the protein standards, the stock dye reagent 
was prepared [500 mg Coomassie Blue was 
dissolved in 500 mL methanol and was added to 100 
mL phosphoric acid and 50 mL double-distilled 
water H2O (ddH2O)] that was diluted in 8 mL 
ddH2O. A total of 2 mL of dye reagent was added to 
each tube of protein standard and was incubated at 
room temperature for at least 5 min. Absorbance of 
the protein standards and experimental samples 
were carried out by the spectrophotometry (Bausch 
and Lomb, Germany) at 595 nm and finally a 
standard curve was plotted (Assady et al., 2011). 
 
Electrophoresis (Li et al., 2013) 
 Areas of the 17cm IPG strips (BIO-RAD) 
were wetted with the above sample in a rehydration 
tray, and mineral oil was added to prevent 
evaporation. The isoelectric focusing (IEF) program 
was set as follows: step 1: 250V (linear) for 30min; 
step 2: 500V (rapid) for 1h; step 3: 4,000V (linear) 
for 4h; step 4: 4,000V (rapid) for 30,000Vh; and 
step 5: 500V (rapid) for 20h (holding step). A total 
HYDATID FLUID PROTEOME 1251
of 2mL of equilibration buffer I [6mol/L urea, 
0.375mol/L Tris-HCL (pH 8.8), 20% glycerol, 2% 
SDS, 2% DTT] was added to the top of the strip in 
an 11-cm equilibration tray, followed by gentle 
rocking for 15min. Equilibration buffer was 
discarded and replaced with equilibration buffer II 
[6mol/L urea, 0.375mol/L Tris-HCL (pH 8.8), 20% 
glycerol, 2% SDS, 2.5% iodoacetamide], followed 
by gentle rocking for 15min. IPG was loaded onto 
an SDS-PAGE gel composed of a 12% separation 
gel.  
 
Gel staining, imaging, and image analysis 
 The proteins were stained using Coomassie 
Brilliant Blue. The image was scanned using Bio-
Rad GM800 Calibrated Densitometer scanner, Spot 
numbers were measured analyzed using Bio-Rad 
PDQuest software, version 6.2.1. 
 
RESULTS 
 
Comparison of protein extraction methods 
 In the untreated sample (Fig. 1A), the host 
serum proteins remain relatively abundant, there 
was no boundaries between protein spots, the gel 
showed obvious streaking and high background. To 
the whole protein extract, that was precipitated 
using ReadyPrepTM 2-D Cleanup Kit (Fig. 1B), 
compared to Figure 1A, the protein spots were more 
clear, and the striations were less. To the whole 
protein extract, that was precipitated using AurumTM 
Serum Protein Mini Kit (Fig. 1C), there was almost 
no protein spots in the map. And to the whole 
protein extract, that was precipitated using TCA-
Acetone (Fig. 1D), there were more single protein 
spots.  
 
Comparison of pH range of IPG strip 
 The molecular weight of most of these 
proteins ranged from 43 to 97kDa and the isoelectric 
points of these proteins ranged from 5 to 9 (Fig. 1). 
Compared with pH3-10 IPG strip, the proteins 
separation was more effective with pH7-10 IPG 
strip (Fig. 2). 
 
Comparison of protein loading amount 
 With 400µg of quantitative loading and IPG 
strips pH 7-10 (Fig. 3D), the proteins spots were 
more evident than with 200µg of quantitative 
loading and IPG strips pH7-10 (Fig. 3C), but the gel 
showed obvious streaking and high background. 
With 400µg of quantitative loading and IPG strips 
pH 3-10 (Fig. 3B), the proteins spots were more 
evident and larger than with 200µg of quantitative 
loading and IPG strips pH 3-10 (Fig. 3A), but the 
proteins spots were less than with 800µg of 
quantitative loading and IPG strips pH3-10 (Fig. 3E). 
Lastly, with 200µg of quantitative loading and IPG 
strips pH 3-10 (Fig. 3E) was observed obvious 
streaking and high background.  
 
DISCUSSION 
 
 Two-dimensional electrophoresis technology 
is the most effective method to isolate the proteins 
of organizations with complex components and is 
one of the main technologies for proteomics 
research. The 2-DE profiles with high resolution and 
good repeatability are prerequisite to analyze and 
identify the proteins of E. granulosus. In this study, 
a series of important factors, such as sample 
preparation, protein quantities, pH range of IPG 
strip and extraction methods, were optimized to 
improve the resolution and repeatability to obtain 
satisfied 2-DE map of hydatid fluid proteome for 
further hydatid fluid proteomics research of E. 
granulosus. 
 HCF proteins are composed of 44% albumin, 
39% α-globulin and β-globulin, and 17% γ-globulin 
(Zhao, 1987), therefore, if the highly abundant 
proteins were not removed, it could interfere the 
detection and analysis for the low abundant proteins. 
When the whole protein extract was precipitated 
using AurumTM Serum Protein Mini Kit, there was 
almost no protein spots in the map, it suggested that 
the low abundant proteins were removed along with 
the highly abundant proteins, so the AurumTM 
Serum Protein Mini Kit was not suitable for 
establish the 2-DE map. Using the TCA Acetone 
Extraction methods, the 2-DE map gets better 
resolution and repetition, indicating that TCA-
Acetone Extraction method is suitable for the 2-DE 
map. Compared with pH3-10 IPG strip, the proteins 
separation was more effective with pH7-10 IPG 
strip   and  it  obtained  more  single  proteins  spots. 
With more proteins quantitative loading, more 
J. LI ET AL.   1252
proteins  spots  were obtained, however, with highly  
     
     
 
 Fig. 1. A comparison of hydatid-cyst fluid protein extracted by different extraction methods  using two-
dimensional electrophoresis from Echinococcus granulosus 
A, Untreated sample; B, Sample was treated using ReadyPrepTM 2-D Cleanup Kit; C, Sample was treated using 
AurumTM Serum Protein Mini Kit; D, Sample was treated using TCA-acetone. 
 
proteins quantitative loading, the gel showed 
obvious streaking and high background and the 
highly abundant proteins were too large to cover 
other important proteins with similar molecular 
weight and isoelectric point, and with lower proteins 
quantitative loading, it was not suitable for detect 
the low abundant proteins which might play an 
important role in the life.  
 In this study, we employed different loading 
amount proteins with 200µg, 400µg, 800µg, and the 
results showed that 2-DE map was more beautiful 
with loading amount proteins with 400µg than 
others. The study showed that the 2-DE profiles 
with high resolution and good repeatability were 
obtained, when the hydatid fluid dealt with 
ReadyPrepTM 2-D Cleanup Kit were analyzed with 
the established 2-DE using 400µg of quantitative 
loading and IPG strips pH7-10. 
 Antigen 5 (Ag5) and antigen B (AgB) were 
identified from E. granulosus hydatid cyst fluid, 
these two antigens are considered the most 
important  antigens  for  the  serodiagnosis of human  
HYDATID FLUID PROTEOME 1253
  
      
 
 Fig. 2. A comparison of hydatid-cyst fluid protein with different pH range of IPG strip using two-dimensional 
electrophoresis from Echinococcus granulosus  
Am pH7-10 IPG strip; B, pH3-10 IPG strip 
 
       
            
 
 Fig. 3. A comparison of hydatid-cyst fluid protein with difference loading amount using two-dimensional 
electrophoresis from Echinococcus granulosus  
A/C, 200µg of quantitative loading; B/D, 400µg of quantitative loading; E, 800µg of quantitative loading. 
J. LI ET AL.   1254
 
CE (Verastegui et al., 1992; Rott et al., 2000; 
Mamuti et al., 2002). AgB is strongly immunogenic 
in patients with echinococcal infections, about 80 to 
90% of serum samples from CE patients and 40% of 
serum samples from AE patients exhibit specific 
antibodies against this antigen (Maddison et al., 
1989; Siles-Lucas and Gottstein, 2001; Mamuti et 
al., 2002). AgB possesses the highest diagnostic 
value among these antigens (Gonzalez-Sapienza et 
al., 2000). 
 The 2-DE in hydatid fluid proteome of E. 
granulosus was successfully established with high 
quality map of separation under the optimization of 
the experimental conditions, which stands as a 
valuable resource for further hydatid fluid 
proteomics research of E. granulosus. 
 
ACKNOWLEDGEMENTS 
 
 This work was supported by grants from the 
National Natural Science Foundation of China (Nº 
30960360).  
 
Conflict of interest declaration 
 The authors declare that there is no conflict of 
interest. 
 
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(Received 20 February 2014, revised 18 June 2014) 
 


Establishment and Optimization of Two-dimensional Electrophoresis Technique in Hydatid Fluid Proteome of Echinococcus granulosus

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Establishment and Optimization of Two-dimensional Electrophoresis Technique in Hydatid Fluid Proteome of Echinococcus granulosus

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