Proteomic analysis of differentially expressed proteins in the three developmental stages of Trichinella spiralis

Accepted Manuscript Title: Proteomic analysis of differentially expressed proteins in the three developmental stages of Trichinella spiralis Author: J.Y. Liu N.Z. Zhang W.H. Li L. Li H.B. Yan Z.G. Qu T.T. Li J.M. Cui Y. Yang W.Z. Jia B.Q. Fu PII: DOI: Reference:

S0304-4017(16)30229-1 http://dx.doi.org/doi:10.1016/j.vetpar.2016.06.021 VETPAR 8054

To appear in:

Veterinary Parasitology

Received date: Revised date: Accepted date:

4-1-2016 13-6-2016 15-6-2016

Please cite this article as: Liu, J.Y., Zhang, N.Z., Li, W.H., Li, L., Yan, H.B., Qu, Z.G., Li, T.T., Cui, J.M., Yang, Y., Jia, W.Z., Fu, B.Q., Proteomic analysis of differentially expressed proteins in the three developmental stages of Trichinella spiralis.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2016.06.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Proteomic analysis of differentially expressed proteins in the three developmental stages of Trichinella spiralis J. Y. Liu a, ‡, N. Z. Zhang a, ‡, W. H. Li a, L. Li a, H.B. Yan a, Z. G. Qu a, T. T. Li a, J. M. Cui a,

a

Y. Yang a, W. Z. Jia a,b, B. Q. Fu a,b

State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of

Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, PR China b

Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease,

Yangzhou, 225009



Corresponding author at: State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, PR China. Tel. +86 931 8342675

Email address: [email protected] ‡ These

authors contributed equally to this work.

Highlights  The life cycle of T. spiralis consist of adult, muscle larvae and newborn larvae.  4,691 proteins were identified by iTRAQ involving in various functions.  1,067 proteins were revealed differently expressed in Ad, ML and NBL.

ABSTRACT

Trichinella spiralis, an intracellular parasitic nematode, can cause severe foodborne zoonosis, trichinellosis. The life cycle of T. spiralis consists of adult (Ad), muscle larvae (ML) and newborn larvae (NBL). The protein profiles in different developmental stages of the parasite remain unknown. In the present study, proteins from lysates of Ad, ML and NBL were identified by isobaric tags for relative and absolute quantitation (iTRAQ). A total of 4,691 proteins were identified in all the developmental stages, of which 1,067 proteins were differentially expressed. The number of up-regulated proteins in NBL was higher than that of the other two groups. The protein profiles from Ad, ML and NBL were compared in pairs. The identified proteins were involved in various functions of T. spiralis life cycle, including sexual maturity, metabolism, utilization of carbohydrates, lipids and nucleotides, and other crucial developmental processes that occur at distinct stages. Further investigation of the transcriptional levels of major sperm protein, serine protease, zinc finger protein, etc. from the different protein profiles using quantitative RT-PCR showed identical results to the iTRAQ analysis. The differentially expressed proteins that are involved in developmental regulation and host-parasite interactions should be further studied.

Keywords: Trichinella spiralis, iTRAQ, adult, muscle larvae, newborn larvae, proteomics

1. Introduction

The intracellular helminth Trichinella spiralis can infect various hosts worldwide (Gottstein et al., 2009), and is of considerable medical and veterinary importance as a cause of the trichinellosis. T. spiralis infections in animals not only cause economic losses for international trade but also remain a serious public risk in developed and developing countries (Kirjušina et al., 2015; Dupouy-Camet, 2000). The life cycle of T. spiralis comprises of adult worm (Ad), muscle larvae (ML) and newborn larvae (NBL), and uniquely, all three stages develop within a single host (Mitreva et al., 2006). The infection begins when ML are ingested, which rapidly enter the intramulticellular niche in the small intestine and develop into Ad through four fast molts. After mating, NBL are born and migrate through the lymph and blood vessels to the skeletal muscle, where they mature into ML. The infected muscle cells transform into nurse cells (Campbell, 1983). The different stages occupy three distinct locations within the host. From the original infection to settlement in the muscle cells, the parasites successively interact with the epithelium of the small intestine, lymph, blood, and finally, muscle cells, and suffer host immune attack as well as different stresses and oxidative environments (Mitreva et al., 2006). T. spiralis has evolved different evasion and suppression mechanisms against these constant pressures and selective forces by expressing different proteins at each stage (Mitreva et al., 2011). Although the expression of stage-specific genes in the helminth has been detected with various immunological, cDNA cloning and RNA sequencing approaches (Liu et al., 2007; Wu et al., 2009; Liu et al., 2012), T. spiralis biology at each developmental stage, including developmental regulation, nutrition metabolism and cell cycle, remains unclear. In this study, we investigated the proteomic profile variations among Ad, ML and NBL of T. spiralis using the isobaric tags for relative and absolute quantitation (iTRAQ) technology, which provided valuable clues and insights to guide future studies on parasite

development.

2. Materials and Methods 2.1. Animals

Specific-pathogen-free (SPF) female Kunming mice, Balb/c mice and Wistar rats were purchased from Lanzhou University Laboratory Animal Center (Lanzhou, China). All animals were handled in strict accordance with the Good Animal Practice requirements of the Animal Ethics Procedures and Guidelines of the People’s Republic of China. This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

2.2. Parasites

The Chinese T. spiralis Henan strain (ISS534) was maintained in Kunming mice. ML were obtained by conventional artificial digestion of infected muscle with pepsin-HCl (1% pepsin, 1% HCl at 42 °C for 45 min) (Liu et al., 2007). After recovery, the ML were used to orally inoculate 100 Balb/c mice and 350 Wistar rats. T. spiralis Ad were collected from the small intestines of the rats three days (Ad3) post-infection (pi) (Liu et al., 2007). Ad recovered from rats 5 days pi were incubated in the culture medium for 24 h to obtain NBL, which were separated from the Ad by filtration through a 25 µm filter (Liu et al., 2007). ML were collected from muscle of Balb/c mice 35 days pi.

2.3. Protein extraction and digestion

The Ad, NBL and ML were collected by centrifugation after three washes. Total protein was extracted in 200 μL lysis buffer, containing 7 M urea, 2 M thiourea (Amersham), 4% CHAPS, 40 mM Tris-HCl (pH 8.5), 2 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM phenylmethanesulfonyl fluoride (PMSF), and then sonicated on ice for 20 min. After reduction by 10 mM dithiothreitol (DTT) at 56°C for 1 h, the proteins were alkylated by 55 mM IAM in the darkroom for 1 h. The treated protein mixtures were precipitated in chilled acetone (4× volume) at -20 °C overnight and centrifuged at 4 °C, 30,000 ╳g for 15 min. Then the pellets were dissolved in 0.5 M TEAB (Applied Biosystems, Milan, Italy) and sonicated on ice. After centrifugation at 30,000 ╳g for 20 min at 4 °C, the supernatant was pipetted for determination of protein concentration by Qubit 2.0® Fluorometer (Invitrogen, USA).

2.4. iTRAQ labeling and strong cation exchange (SCX) fractionation

The peptide-labeled iTRAQ was performed as previously described (Sardar et al., 2013; Lynn et al., 2013). Briefly, after digestion by Trypsin Gold (Promega, Madison, WI, USA), the peptides were freeze-dried in a vacuum. Then, the peptides were reconstituted in 0.5 M TEAB and labeled according to the manufacturer’s protocol for 8-plex iTRAQ reagent (Applied Biosystems). The samples from ML were labeled with iTRAQ tags 114. Ad samples were labeled with iTRAQ tags 115. NBL samples were labeled with iTRAQ tags 117. All samples were incubated at room temperature for 2 h. The labeled peptide mixtures were then pooled and dried by vacuum centrifugation. SCX chromatography was performed in the LC-20AB High Performance Liquid Chromatography (HPLC) pump system (Shimadzu, Kyoto, Japan). The iTRAQ-labeled peptide mixtures were dissolved in 4 mL buffer A (25 mM NaH2PO4 in 25% ACN, pH 2.7) and loaded onto a 4.6×250 mm Ultremex SCX column containing 5 μm particles

(Phenomenex). The peptides were eluted as previously described (Sardar et al., 2013) with a gradient of buffer A and buffer B (25 mM NaH2PO4, 1 M KCl in 25% ACN, pH 2.7). The eluted peptides were pooled into 20 fractions, desalted with a Strata X C18 column (Phenomenex) and vacuum-dried.

2.5. LC-ESI-MS/MS analysis

The final concentration of peptides was adjusted to 0.5 μg/μL on average, and 10 µL was loaded onto a 2 cm C18 trap column and then eluted onto a 10 cm analytical C18 column (inner diameter 75 µm) on LC-20AD nano HPLC (Shimadzu, Kyoto, Japan). The samples were separated by gradient Buffer B from 2 to 80% and finally returned to 5% in 1 min. The peptides were subjected to nanoelectrospray ionization followed by tandem mass spectrometry (MS/MS) in Q EXACTIVE (Thermo Fisher Scientific, San Jose, CA) coupled online to the HPLC. The MS/MS was performed as previously described (Lynn et al., 2013).

2.6. Protein data analysis

The raw MS data files were converted using Proteome Discoverer 1.2 software (PD 1.2, Thermo). Protein identification was performed using the Mascot software Matrix 2.3.02 (Science, London, UK) against the T. spiralis database downloading from UniProt of EMBL. The differentially expressed proteins were defined as those with 1.5 fold change relative to one another, with p < 0.05. The gene name, functional annotations, protein orthologous classification, molecular interaction and reaction networks were analyzed by Blast, Gene Ontology and KEGG Pathway, respectively.

2.7. Quantitative real-time RT-PCR verification

Total RNA of T. spiralis Ad, NBL or ML were extracted using Trizol reagent (Invitrogen, USA), as per the manufacturer’s instructions. RNAs were dissolved in RNase-free ddH2O (TaKaRa, China) and reverse transcribed first-strand cDNAs were used as templates for realtime PCR. The SYBR Green-labelled primers (Table 1) were synthesized by TaKaRa (Dalian, China). The RT-PCR was performed on ABI PRISM® 7500 Sequence Detection System (Applied Biosystems). GAPDH was used as a housekeeping gene. The differences in qPCR quantitation were compared with two-way ANOVA.

3. Results

3.1. Primary data analysis and protein detection

The spectra generated from the iTRAQ experiment matched 4,691 proteins through BLAST in GenBank and SWISS-PORT. The differentially expressed proteins (DEPs) in the three developmental stages were analyzed using pair-wise (NBL versus Ad, Ad versus ML, and NBL versus ML) comparison of the identified spectra. A total of 1,067 proteins were differentially expressed between two arbitrary stages. The number of DEPs between NBL and Ad3 was slightly less than that in ML vs Ad3 and ML vs NBL. As shown in Fig. 1, a total of 146 proteins were found to be differentially expressed among the pair-wise comparisons. The number of stage-specific proteins were highest in ML vs NBL (352 proteins), which was threefold more than that of NBL vs Ad3 (93 proteins).

3.2. Identification of DEPs in three developmental stages of T. spiralis

DEPs in the three developmental stages of T. spiralis were involved in diverse functions in the life cycle, including regulation of worm growth and sexual maturity (e.g. major sperm protein), metabolism and utilization of carbohydrates, lipids and nucleotides (e.g. glucose-6phosphate isomerase), modulation of host cell functions for the parasite benefit via excretorysecretory (E-S) antigens (e.g. serine protease), and other crucial developmental processes that occur at distinct stages.

3.3. Functional classification of the identified proteins

The possible functions of the identified proteins were predicted by the gene function classification system GO, which has three ontologies including molecular function (16,015 proteins), cellular component (8,740 proteins) and biological process (4,676 proteins). Thereinto, numbers of proteins were predicted by multiple functions. The main biological functions of the proteins were: cellular process (2,436 proteins, 15.21%), metabolic process (2,157 proteins, 13.47%) and single-organism process (1,755 proteins, 10.96%). The proteins mainly composed cell and cell components (3,899 proteins, 44.61%), but few composed extracellular matrices and regions (58 proteins, 2.44%). The percentages of proteins classified into bonding and catalytic activities were the highest among molecular functions, which were up to 82.42%. The GO analysis of the identified proteins is shown in Fig. 2. Among the DEPs, several significantly expressed proteases involved in glycolysis pathway, redox system and other important biological processes were selected, as listed in Table 2. Clusters of Orthologous Groups of proteins (COG) classification in the three developmental stages revealed that the proteins were primarily involved in energy production and conversion, amino acid transport and metabolism, carbohydrate transport and

metabolism, lipid transport and metabolism, translation, ribosomal structure and biogenesis, transcription, replication, recombination and repair, post-translational modification, protein turnover, chaperones, signal transduction mechanisms, intracellular trafficking, and secretion (Fig. 3). The results indicated that proteins from the three developmental stages were involved in every aspect of parasite growth and metabolism.

3.4. Pathway enrichment of identified proteins

The molecular interaction and reaction networks of identified proteins were further analyzed through KEGG pathway maps. The results showed that the pathways including metabolism, lysosome, focal adhesion, spliceosome, RNA transport, endoplasmic reticulum processing were enriched more than 100 proteins. The pathways involved in reproductive development, nutrition metabolism, growth, apoptosis and antioxidation were identified in each pair-wise comparison.

3.5. Quantitative real-time RT-PCR analysis validation of DEPs

Eight genes from DEPs designated E5S8I3 and E5SED3 (major sperm protein, gene locus tag, Tsp_00052 and Tsp_02102), B7SIW4 (serine protease gene locus tag, Tsp_SP-1.2), Q27071 (53kDa E-S antigen, gene locus tag, Tsp_gp53), E5SF13 (zinc finger protein, gene locus tag Tsp_02330), E5RY73 (histone H2B subunit, gene locus tag, Tsp_07127), E5S882 (DNA polymerase ε subunit, gene locus tag, Tsp_07920), and E5S6R9 (neural proliferation differentiation and control protein, gene locus tag, Tsp_07015) were selected for qRT-PCR analysis to quantify their transcriptional levels (Fig. 4). The results revealed that the genes encoding major sperm protein, histone H2B subunit and DNA polymerase ε subunit were

significantly higher expressed in the Ad3 than in the other two developmental stages. The 53 kDa E-S antigens were up-regulated in ML and Ad3 than in NBL. No significant differences in transcript levels of the zinc finger protein and neural proliferation differentiation and control protein were detected among the three developmental stages. The qPCR results were mainly consistent with the iTRAQ analysis (listed in Table 3), which suggested that the proteomics analysis tool used in this study is reliable.

4. Discussion

Numerous proteins are involved in differential regulation of distinct life-cycle stages of T. spiralis. The differentially expressed genes at each developmental stage have been identified in previous studies (Liu et al., 2007; Liu et al., 2012). However, the protein profiles in the three developmental stages remain largely unknown. Herein, we applied a quantitative proteomics approach with iTRAQ technique to fully reveal the protein expression profiles at Ad3, ML and NBL. A total of 4,691 proteins were identified in all three developmental stages, involved in crucial biological processes, such as growth, aging, metabolism, adhesion, recombination and repair. Among the 4,691 identified proteins, 22.8% were differentially expressed, of which more than one-third were differentially expressed in Ad3. This was in contrast to a previous study that identified more transcripts in the NBL (Liu et al., 2012). The Ad worms undergo the processes of maturation to reproductive stage within the intestinal epithelium, and need to resist oxidation and modulate host immune responses (Robinson et al., 2010). More functional proteins should be expressed to accurately regulate various biological processes. Serine proteases were shown to be involved in the invasion of mammalian host cells (Dzik, 2006; Nagano et al., 2003), and are up-regulated in larvae after activation by bile (Liu et al.,

2013; Ren et al., 2013). Here, four homologous serine proteases were differentially expressed in the Ad3, and more functions of the proteins should be investigated. Further comprehensive analysis of the high-level functions of proteins in the three developmental stages in T. spiralis by KEGG showed that all the identified proteins were predicted to be involved in 250 pathways. The oocyte meiosis is a specialized type of cell division in which one round of DNA replication is followed by two meiotic divisions to produce four daughter cells (MacLennan et al., 2015; Jessberger, 2012; Page et al., 2003). Each cell contains half the number of chromosomes as the original parent cell (Herbert et al., 2015; Baudat et al., 2013). The meiotic oocyte is a continuous process from fetus to adult, which is divided into meiosis I and meiosis II phases (MacLennan et al., 2015). During fetal and infant development, sister chromatid is exchanged between homologous chromosomes until the synaptonemal complex disassembles (Jessberger, 2012; Page et al., 2003). In adult oocytes, the homologous chromosomes are disjunctive by reductional division during meiosis I (MI) and the sister chromatids are equational segregation during meiosis II (MII) (Baudat et al., 2013; Petronczki et al., 2003). Here, several DEPs were enriched in each pair-wise comparison (NBL versus Ad, Ad versus ML, and NBL versus ML), and six up-regulated proteins and one down-regulated protein were detected in ML as compared to Ad3, but the regulatory mechanism is unclear. The results were in contrast to a previous study that showed that most differentially expressed genes related to oocyte meiosis were up-regulated in Ad as compared to the other two stages (Liu et al., 2012). This difference may result from splice variants, protein process, as well as the different sequencing method and data library used. The processes of oocyte meiosis in T. spiralis developmental stages need further study. The metabolism of carbohydrate (typically glucose) is one of the main biochemical processes for providing energy that is temporarily stored in the cells in the form of ATP. In this study, the key enzymes, fructokinase and lactate dehydrogenase, involved in anaerobic metabolism

were significantly up-regulated in ML as compared to the other two stages, which confirms that anaerobic metabolism is more activated in ML stage (Agosin et al., 1959; Janssen CS, et al., 1998). Thereinto, lactate dehydrogenase can catalyze the conversion of pyruvate into lactate, the end product of glycolysis. However, acetic, valeric and caproic acids but not lactic acid are the major end products of glycolysis (Agosin et al., 1959). Here, phosphoenolpyruvate carboxykinase, the crucial enzyme in gluconeogenesis, was also upregulated in ML as compared to NBL and Ad3, which indicated maximal activation of gluconeogenesis in ML. Alcohol dehydrogenase was detected in the three developmental stages of T. spiralis in this study, but not in homogenates of T. spiralis larvae in a previous report (Agosin et al., 1959). Helminths have developed various protective antioxidant-related molecules against the potentially damaging effects of reactive oxygen species (ROS) derived from normal aerobic metabolism and activated leukocytes in the mammalian host (Salinas et al., 1998; HenkleDührsen et al., 2001). When the parasites undergo rapid growth phases and produce numerous off-springs, the redox systems are usually most activated (Robison et al., 2010). In this study, higher levels of peroxiredoxins (PRXs), thioredoxin peroxidases (TPXs), superoxide dismutase (SOD), and glutathione peroxidases (GPXs) were detected in Ad3, which suggested that the parasite encounters an environment of high oxidative stress at this stage. PRX enzymes catalyze the reduction of hydrogen peroxide (H2O2) to H2O (Chae et al., 1994). The TPXs are 2-Cys PRXs, which can reduce H2O2 by electron transport via the thioredoxin system (comprised of thioredoxin, thioredoxin reductase and NADPH) (Kang et al., 1998; Gretes et al., 2012). SOD catalyzes the dismutation of superoxide anion into H2O2 and oxygen (Southorn et al., 1988; Sies, 1993). GPXs can catalyze the reduction of H2O2 by oxidizing glutathione (GSH) (Henkle-Dührsen et al., 2001). These enzymes are major contributors to

self-protection of the parasite during invasion, migration and reproduction. In this study, the antioxidant enzymes were enriched in all three developmental stages. In conclusion, a total of 4,691 proteins were identified by iTRAQ technique in the three developmental stages, and 1,067 proteins were differentially expressed among pair-wise comparisons (NBL versus Ad, Ad versus ML, and NBL versus ML) in this study. GO analysis revealed that some proteins are involved in important functions related to cellular, metabolic and single-organism processes. The identification of these proteins in NBL, ML and Ad3 has facilitated better understanding of the developmental mechanism of T. spiralis and the hostparasite interactions.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

This study was funded by the National Natural Science Foundation of China (No. 31272555) and partially supported by the Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006).

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Figure legends:

Figure 1. Distribution of the 1067 differentially expressed genes between the three stages.

Figure 2. Classification of protein functions by Gene Ontology (GO). Proteins were classified as three ontologies including molecular function, cellular component and biological process. A total of 16,015 proteins were predicted to participate in molecular functions, 8,740 proteins in cellular components and 4,676 proteins in biological processes.

Figure 3. COG coverage of the protein sequence. A total of 24 groups of differentially expressed proteins were clustered by orthologous groups.

Figure 4. Confirmation of differentially expressed genes of Trichinella spiralis by qRT-PCR. Comparison of the relative quantitation of each gene expressed in ML, NBL and Ad3 in pairs. ** P < 0.01, *** P < 0.001, NS: not significant.

Table 1 Primers for verification of mRNA transcriptional levels of genes identified by iTRAQ from Trichinella spiralis

Gene name

Access

Primer sequence

number major sperm protein

Tsp_02102

PF：5’-CGCACCAATTCCAGAAAACT-3’ PR：5’-GGTTCACCTTGAGCCCTGTA-3’

major sperm protein

Tsp_00052

PF：5’- CCGCTACGTGGTCTTACCAT-3’ PR：5’- CCCATGACGAACCAATCTTC-3’

serine protease gene

Tsp_SP-1.2

PF：5’-CCTCCCAAGTTGATTCAGGA -3’ PR：5’-ATGAATCAGGAAGCCACCAC-3’

53kDa excretory/secretory antigen

gp53 gene

PF：5’-TGATACGGACGATGCTTACG-3’ PR：5’-TGTTGGAAAAACCCTTTTGG-3’

DNA polymerase ε subunit

Tsp_07920

PF：5’-GGGCAACTTCACAACAGCTT-3’ PR：5’-GCTCCGTGAAGTTCCATAGG-3’

histone H2B subunit

Tsp_07127

PF：5’-GAAGCAAGTCCATCCCGATA-3’ PR：5’-TTCACTAACAGCGTGCTTGC-3’

Neural proliferation differentiation Tsp_07015

PF：5’-ACCGAGGTCAGAGTGGATAT-3’

and control protein

PR：5’-ATGACGGTGGATTGACGAAC-3’

zinc finger protein

Tsp_02330

PF：5’-CGTATGGATCTGGCACTCCT-3’ PR：5’-GGAAGTCCGTCAATTTGCTC-3’

reduced glyceraldehyde-phosphate

PF ： 5’-AGATGCTCCTATGTTGGTTATGGG-

dehydrogenase (GAPDH)

3’ PR：5’- ACTGTCTTTTGGGTTGCCGTT-3’

Table 2 Details of differentially expressed proteases in adults, muscle larvae and newborn larvae of Trichinella spiralis. P value

Ratio Accession

Description

Coverage

Unique Peptides

Ad3/ML

NBL/Ad3

Ad3/ML

NBL/Ad3

Significance

Significance

B7SIW5

Serine protease

19.0

6

0.15

0.12

2.60E-19

1.21E-17

B7SIW4

Serine protease

28.1

5

0.13

1.53

5.95E-22

4.39E-21

B7SIW3

Serine protease

18.8

8

0.13

0.20

7.12E-22

3.35E-11

B0F9U0

Putative serine protease

59.4

9

0.31

0.20

2.34E-8

6.90-11

E5SEC2

6-phosphofructokinase

20.9

14

0.64

0.54

0.034

0.011

11.0

3

0.54

0.41

0.0038

0.00024

E5SNL6

Malate/L-lactate dehydrogenase subfamily

E5SLA7

Phosphoenolpyruvate carboxykinase

78.8

46

0.76

0.61

0.20

0.045

E5S643

Alcohol dehydrogenase

22.2

9

0.80

0.76

0.28

0.27

E5SNM2

Peroxiredoxin TSA1

6.5

1

1.20

0.97

0.43

0.91

E5RZT8

Superoxide dismutase

58.9

10

0.65

0.60

0.042

0.036

E5S8F5

Thioredoxin peroxidase 1

51.2

10

0.82

0.67

0.35

0.010

E5SEV5

Glutathione peroxidase

72.1

7

0.70

0.66

0.086

0.089

Table 3 Details of selected differentially expressed proteins in adults, muscle larvae and newborn larvae of Trichinella spiralis used in qRT-PCR.

Acces sion

Q270 71 B7SI W4 E5S88 2

E5S6 R9

E5SF 13

E5SE D3 E5RY 73 E5S8I 3

Description

53kDa excretory/se cretory antigen Serine protease DNA polymerase epsilon subunit 2 Neural proliferation differentiati on and control protein 1

Zinc finger protein 364

Major sperm protein Histone H2B Major sperm protein

Ratio

Cover age

Uniq ue Pepti des

Pfam IDs

52.2

4

28.1

P value NBL/M L Signific ance

Ad3/ ML

NBL/ ML

NBL/ Ad3

Ad3/M L Signific ance

-

0.11

1.51

0.07

1.47E24

0.22

4.80E27

5

Pf000 89

0.13

1.53

1.53

5.95E22

0.21

4.39E21

43.5

3

-

0.53

0.87

0.30

0.0023

0.622

8.74E07

11.0

3

Pf068 09

0.52

2.65

0.42

0.0023

0.004

0.00033

28.1

6

Pf000 97; Pf126 78; Pf128 61

1.13

0.91

1.16

0.60

0.75

0.61

65.2

6

Pf006 35

2.88

0.38

2.96

3.25E06

0.00074

0.00011

65.0

1

Pf001 25

3.63

3.34

4.75

1.52E08

0.00038

2.83E08

28.5

1

Pf006 35

9.41

0.13

9.35

6.92E23

9.93E13

1.66E15

23

NBL/A d3 Signific ance