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Fish & Shellfish Immunology 30 (2011) 324e329
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Fish & Shellfish Immunology
journal homepage: www.elsevier.com/locate/fsi
Molecular cloning, characterization and expression analysis of macrophage
migration inhibitory protein (MIF) in Chinese mitten crab, Eriocheir sinensis
Wei-Wei Li, Xing-Kun Jin, Lin He, Ying Wang, Li-Li Chen, Hui Jiang, Qun Wang*
School of Life Science, East China Normal University, North Zhong-Shan Road, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 September 2010
Received in revised form
21 October 2010
Accepted 4 November 2010
Available online 16 November 2010
Macrophage migration inhibitory factor (MIF) as a multi-functional cytokine mediating both innate and
adaptive immune responses, however, their function within the innate immune system of invertebrates
remains largely unknown. Therefore, we investigated the immune functionality of MIF in Chinese mitten
crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The fulllength MIF cDNA (704 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a E. sinensis cDNA library. The MIF cDNA contained a 363 bp open reading frame (ORF) that
encoded a putative 120 amino acid (aa) protein. Comparisons with other reported invertebrate and
vertebrate MIF sequences revealed conserved enzyme active sites. MIF mRNA expression in E. sinensis
was (a) tissue-specific, with the highest expression observed in hepatotpancreas, and (b) responsive in
hemocytes, hepatopancreas and gill to a Vibrio anguillarum challenge, with peak exposure observed 8 h,
12 h and 12 h post-injection, respectively. Collectively, data demonstrate the successful isolation of MIF
from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Eriocheir sinensis
Macrophage migration inhibitory factor
mRNA expression profile
Real-time PCR
1. Introduction
Since found first in 1966 [1,2], the mammalian macrophage
migration inhibitory factor (MIF), has been shown to correlate with
the regulation of macrophage functions [3,4], lymphocyte immunity
[5,6], endocrine function [7e11] and a number of immune and
inflammatory diseases [7,8,12e22]. MIF initially identified as an
inhibitor of random migration of macrophages, has now emerged to
be a multi-functional factor involved in immune response, glucose
and lipid metabolism. In addition, MIF can modulate glycolysis and
insulin resistance in insulin target cells including adipocytes and
myocytes [6].
MIF was constitutively expressed by a variety of immune and
some non-immune cells and released into the circulation from
preformed intracellular stores by a non-conventional leaderless
pathway [7,23,24]. As one of the first lymphocyte-derived cytokines, MIF was identified as a soluble factor produced by antigenactivated T lymphocytes that inhibited the random migration of
macrophages [1,2,25]. Furthermore, recent research shows a more
prominent role of MIF as a multi-functional cytokine mediating
both innate and adaptive immune responses. To data, mammalian
MIF is an immunomodulator that controls macrophage functions,
* Corresponding author. Fax: þ86 21 62233754.
E-mail address: [email protected] (Q. Wang).
1050-4648/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fsi.2010.11.008
resulting in the promotion of proinflammatory cytokine expression
(TNF-, IL-1, IL-2, IL-6, IL-8, and IFN-) [3,5,20], nitric oxide (NO)
release [26] and COX-2 activity [27]. In activated macrophages, MIFinduced TNF- leads to further MIF release, resulting in optimal
expression of TNF- by macrophages [3]. MIF also up-regulates the
expression of Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide (LPS) and induces the activation of monocytes/
macrophages, suggesting potential involvement of MIF in innate
immune responses [28]. Homologues of mammalian MIF have been
detected in chicken, fish, ticks, nematode and even plant [29]. All
evidences available up to now imply that MIFs might exert
important and similar functions among different species.
Chinese mitten crab, Eriocheir sinensis, is an economically
important freshwater species for aquaculture in China. However,
bacterial- and viral-born disease have blossomed within booming
E. sinensis cultures, incurring catastrophic losses to crab aquaculture [30]. Crab, as well as other invertebrates, do not possess an
adaptive immune system and must rely on efficient innate immune
defenses [31]; therefore, obtaining a better understanding of the
innate immune ability of crab and their defense mechanisms has
become a research priority. In order to determine the participation
of MIF in innate immune responsiveness in crustaceans, and
identify key mechanistic steps for improving immunity and associated mortality in aquaculture E. sinensis, we (1) cloned the fulllength Es-MIF cDNA using EST sequences identified from a E.
sinensis hepatopanreatic cDNA library previously constructed by
W.-W. Li et al. / Fish & Shellfish Immunology 30 (2011) 324e329
our laboratory [32], (2) examined mRNA tissue-dependent
expression patterns, and (3) determined the temporal response of
Es-MIF expression in response to an immune challenge via Vibrio
anguillarum exposure.
2. Method and materials
325
Table 1
Primer sequences.
Primers
Primers for 50 RACE PCR
MIF 50 first GSP primer
MIF 50 nested GSP primer
Sequences
Code
50 -GCTCTGATTTGC
CAAGCATCTCACTA30
50 -GGAACATTTGTG
AACACCTCCAGCA30
GSP-50 -f
50 -GATGGTGGACAA
TCGGCTGTGGe30
50 -TTTCCATGCAATA
CTTGGCCGCTGAA30
GSP-30 -f
GSP-50 -n
2.1. Sample preparation
Healthy E. sinensis (mean weight ¼ 100 g) were collected from
the Tongchuan aquatic product market in Shanghai, China. Crabs
were placed in an ice bath for 1e2 min until lightly anesthetized
prior to sacrifice. The following tissues were harvested, snap frozen
in liquid nitrogen, and stored at 80 C until nucleic acid analysis,
hepatopancreas, brain, gills, intestine, hemocytes, muscle, stomach,
heart, testis and ovarian. For cloning and expression analysis,
tissues from 10 crabs were pooled and ground with a mortar and
pestle prior to extraction.
Primers for 30 RACE PCR
MIF 30 first GSP primer
MIF 30 nested GSP primer
ClontechÔ Kit primesr
Universal primer A mix
Nested universal primer A
2.2. Nucleic acid extraction
Total RNA was extracted from E. sinensis using TrizolÒ reagent
(Invitrogen) according to the manufacturer’s protocol. The
concentration and quality of total RNA were estimated by spectrophotometry (absorbance at 260 nm) and agarose-gel electrophoresis, respectively.
2.3. First-strand cDNA synthesis
Total RNA (5 mg) isolated from hepatopancreas was reverse
transcribed using the SMARTÔ cDNA kit (Clonetech) for cDNA
cloning. For expression analysis, total RNA (4 mg) was reverse
transcribed using the PrimeScriptÔ RT-PCR Kit (TaKaRa) for semiquantitative RT-PCR analysis or the PrimeScriptÔ Real-time PCR Kit
(TaKaRa) for real-time quantitative RT-PCR (qRT-PCR) analysis.
2.4. The cDNA full-length clone by RACE
The Es-MIF cDNA sequence was extended using 50 and 30 RACE
and a total of four gene-specific primers (Table 1) based upon the
original EST sequence (as described above; GenBank accession no.
GE340285). The initial 30 RACE PCR reaction was carried out in
a total volume of 50 ml that contained 2.5 ml of the first-strand cDNA
reaction as template, 5 ml of 10 Advantage 2 PCR buffer, 1 ml of
10 mM dNTPs, 5 ml of 10 mM gene-specific primer (GSP-30 -f and
GSP-30 -n; Table 1), 1 ml of Universal Primer A Mix (UPM; Clonetech,
USA), 34.5 ml of sterile deionized water, and 1 U 50 Advantage 2
polymerase mix (Clonetech). Diluted products of the first PCR 30
RACE reaction (1:50 with TricineeEDTA buffer) served as template
for the 30 nested PCR reaction. With the exception of the template
and nested primer (NUP), contents of the nested PCR reaction were
identical to the initial reaction. For 50 RACE, SMARTÔ cDNA kit UPM
and NUP were used as forward primers in initial and nested PCR
reactions in conjunction with the reverse gene-specific primers
GSP-50 -f and GSP-50 -n, respectively (see Table 1 for sequence). PCR
amplification conditions for initial 30 and 50 RACE were as follows: 5
cycles at 94 C for 30 s, 72 C for 3 min; 5 cycles at 94 C for 30 s,
70 C for 30 s, and 72 C for 3 min; 20 cycles at 94 C for 30 s, 68 C
for 30 s, and 72 C for 3 min. Nested PCR amplification conditions
were as follows: 20 cycles at 94 C for 30 s, 68 C for 30 s, and 72 C
for 3 min. PCR amplicons were size separated and visualized on an
ethidium bromide stained 1.2 % agarose gel. Amplicons of expected
sizes were purified with E.Z.N.AÒ Gel Extraction Kit (Omiga BioTek),
and inserted into a pTZ57RÒ Vector (Promega). Positive clones
GSP-30 -n
50 -CTAATACGACTCACT
ATAGGGCAAGCAGTGG
TATCAACGCAGAGT-30
50 -CTAATACGACTCA
CTATAGGGC-30
50 -AAGCAGTGGTAT
CAACGCAGAGT-30
Primers for RT-PCR and Real-time PCR analysis
MIF 50 primer
50 -TGACTTGTTTTC
CTCCACTCCC-30
MIF 30 primer
50 -GGTGTTCACAA
ATGTTCCCAAGG-30
b-actin 50 primer
50 -CTCCTGCTTGCT
GATCCACATC-30
b-actin 30 primer
50 -GCATCCACGAG
ACCACTTACA-30
M-RT-R
M-RT-F
b-RT
b-FT
containing inserts of the expected size were sequenced using T7
and SP6 primers.
2.5. Sequence analysis
Es-MIF full-length cDNA and deduced amino acid sequences
were compared with other sequences reported in NCBI’s GenBank
using the BLAST program. MIF cDNA and deduced amino acid
sequences from E. sinensis and representative vertebrates and
invertebrates were compared by multiple sequence alignment
using ClustalX. Anphylogenetic treewas constructed with MEGA4.0.
E. sinensis MIF cDNA and deduced amino acid sequence were
deposited under GenBank accession numbers HM775084.
2.6. Immune challenge and hemocyte isolation
Chinese mitten crabs (n ¼ 155; 70 5 g wet weight) were
acclimated for 1 week at 20e25 C in filtered, aerated freshwater
prior to injection with approximately 70 ml live V. anguillarum
resuspended in 0.1 mol L1 PBS (pH ¼ 7.0, 109 CFU mL1) or a vehicle
control of PBS (pH ¼ 7.0). Five crabs were randomly selected 2, 4, 6, 8,
12, 16, 32 and 48 h post-injection from experimental and control
groups for hemolymph collection using a syringe (approximately
2.0 ml per crab). An equal volume of anticoagulant solution was then
added to each hemolymph sample, and centrifuged at 800 g at 4 C
to isolate hemocytes. Hemocytes were stored at 80 C after addition of 1 mL Trizol reagent (Invitrogen) for subsequent RNA
extraction.
2.7. Real-time qRT-PCR analysis
The mRNA expression of MIF was measured by real-time RTPCR. Briefly, total RNA was isolated from hepatopancreas, gill, ovary,
testis, muscle, heart, brain, stomach, intestine and hemocytes of
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W.-W. Li et al. / Fish & Shellfish Immunology 30 (2011) 324e329
2 SYBR Premix Ex Taq (TaKaRa), 0.5 ml diluted cDNA template,
11.0 ml PCR-Grade water, and 0.5 ml of each primer. PCR conditions
were as follows, 95 C for 30 s; followed by 40 cycles of 95 C, and
a 0.5 C/5 s incremental increase from 60 C to 95 C that lasted 30 s
per cycle. Resultant data was analyzed using the CFX ManagerÔ
software (Version 1.0).
2.8. Statistical analysis
Statistical analysis was performed using SPSS software (Ver11.0).
Data represent the mean standard error (S.E.). Statistical significance was determined by one-way ANOVA [33] and post hoc
Duncan multiple range tests. Significance was set at P < 0.05.
3. Results
3.1. Cloning and characterization of Es-MIF cDNA
The full-length Es-MIF cDNA sequence cloned from E. sinensis
hepatopancreas was 704 bp long and contained a 363 bp ORF that
encoded a 120 amino acid protein, a 103 bp 50 UTR, and a 235 bp 30
UTR (Fig. 1).
3.2. Amino acid sequence alignment
Alignment of the Es-MIF amino acid sequence with those
reported for other organisms demonstrated conservation of sites
with demonstrated catalytic activity (Fig. 2). Overall homology was
high among other species in reference to Es-MIF, with Maconellicoccus hirsutus and Acyrthosiphon pisum exhibiting the greatest
percent identity (56%), followed by Bombyx mori (52%), Ascaris suum
(46%), Amblyomma americanum (46%) and Xenopus tropicalis (45%).
3.3. Phylogenetic analysis of Es-MIF
Fig. 1. E. sinensis MIF cDNA and deduced amino acid sequences. The triad of conserved
catalytic active sites (P2, K33 and Cys57) are shaded.
unchallenged crabs (used as a pool) and hemocytes, hepatopancreas and gill of bacteria-challenged crabs (used as individually).
Real-time qRT-PCR was conducted using the CFX96Ô Real-Time
System (Bio-Rad). Gene-specific primers (Table 1) were designed
based upon the cloned MIF cDNA to produce a 168 bp amplicon.
Samples were run in triplicate and normalized to the control gene
b-actin, Es-MIF expression levels were calculated by the 2DDCt
comparative CT method. Real-time qPCR amplification reactions
were carried out in a final volume of 25 ml, which contained 12.5 ml
Phylogenetic analysis of Es-MIF from representative crustaceans, mammals, and pisces (Fig. 3) produced an NJ-phylogenetic
tree, that contained two distinct branches, suggesting a phylogenetic relationship and shared common ancestor among MIF genes
in different species. The first branch was comprised by vertebrate
including mammals and fish, the second branch was composed by
invertebrate include E. sinensis, which supporting traditional
taxonomic relationships.
3.4. Tissue distribution of Es-MIF expression
As determined by Real-time qRT-PCR, detectable Es-MIF
expression was widely observed in the hepatopancreas, hemocytes,
gill, brain, muscle, heart, intestine, stomach testis and ovarian of E.
Fig. 2. Multiple alignment of MIF amino acid sequences. Identical (*) and similar (. or :) amino acid residues are indicated. Gaps () were introduced to maximize the alignment.
Protein abbreviations and corresponding GenBank accession numbers are as follows: Eriocheir sinensis (HM775084); Homo sapiens (CAG30406.1); Danio rerio (NP_001036786.1);
Mus musculus (AAA91638.1); Ornithorhynchus anatinus (XP_001507338.1); Salmo salar (NP_001117081.1); Cyprinus carpio (ABY71027.1); Oncorhynchus mykiss (ACO08446.1); Tetraodon nigroviridis (AAW50793.1); Branchiostoma belcheri tsingtauense (AAT77698.1); Sus scrofa (NP_001070681.1); Bos taurus (NP_001028780.1); Ovis aries (NP_001072123.1);
Rattus norvegicus (AAB04024.1); Acyrthosiphon pisum (NP_001156107.1); Bombyx mori (NP_001040199.1); Ascaris suum (49257069); Trichuris trichiura (5327286); Strongyloides ratti
(198448303).
W.-W. Li et al. / Fish & Shellfish Immunology 30 (2011) 324e329
Fig. 3. Neighbor-joining phylogenetic tree of MIF amino acid sequences reported in
representative taxa. See Fig. 2 for GenBank accession numbers.
sinensis (Fig. 4). Expression was highest in hepatopancreas, and
comparable among heart, gill, intestine and ovarian.
3.5. Temporal expression of Es-MIF in immune challenged
hemocytes
Es-MIF expression level, as measured by real-time qRT-PCR, was
induced in hemocytes, hepatopancreas and gill following exposure
to V. anguillarum (Fig. 5 and Fig. 6). Es-MIF expression in hemocyte
was significantly greater than the vehicle control after 4 h, 6 h, 8 h
and 12 h post V. anguillarum stimulation (P < 0.05). Es-MIF
expression in hemocyte was up-regulated at 4 h post-injection, and
peaked to 16.5-fold that of the vehicle control after 8 h, and then
decreased to levels with no significant different with the vehicle
16 h post-injection (Fig. 5). The expression of Es-MIF after immune
challenge was determined with real-time PCR in both hepatopancreas and gill tissues in E. sinensis (Fig. 6). Es-MIF mRNA levels in
hepatopancreas and gill increased sharply 12 h after exposure to V.
anguillarum, and decreased down after 16 h post-exposure. Control
reactions, in which were induced with PBS, yielded no significant
increase in expression levels.
4. Discussion
327
Fig. 5. Temporal MIF mRNA expression in response to V. anguillarum challenge (black
bars). Hemocytes collected from crabs injected with V. anguillarum (black bars) or
vehicle (white bars) were compared with respect to MIF mRNA expression (relative to
b-actin) using Students t-tests. Bars represent mean S.E. (n ¼ 6). Statistical significance is indicated with an asterisk (P < 0.05).
protozoa, which may result from its functional significance.
Correlation between mechanism of action and role in diseases
reveals that MIF is in a cytokine network not only as an effector
molecular but also as a proinflammatory mediator and serves as
a potential therapeutic target [34]. The presence of Es-MIF in this
study further demonstrated that MIF is evolutionarily conserved,
The amino-terminal proline residue (Fig. 1, position P) which is
crucial for the catalytic activity of isomerase [35e39] was found in
Es-MIF (shown in Fig. 1). In addition, the invariant lysine residue
(Fig. 1, position K) observed in many species include mammalian,
which contributes to the isomerase activity of the protein [38], is
also present in Es-MIF. Because crucial amino acid residues, thus we
expected Es-MIF to have the same activity as mammalian MIF.
Mammalian MIF is distinguished by the presence of three
conserved cysteines (Fig. 1, Cys57, Cys60, and Cys81), the first two of
which define a CXXC motif that mediates thiol protein oxidoreductase activity [35e39]. Except that Cys is conserved in Es-MIF,
the latter two cysteines as well as the CXXC motif, are absent in EsMIF, and that is the same with the report in Haliotis diversicolor
supertexta MIF [40]. Therefore, whether the absence of two cysteines as well as the CXXC motif has any effect on the function of ESMIF needs to be further studied.
MIF was found to be conserved throughout species, from both
invertebrates and vertebrates, even in single-celled parasitic
Fig. 4. Tissue-dependent MIF mRNA expression in E. sinensis. MIF mRNA expression in
hemocytes, heart, hepatopancreas, gill, stomach, muscle, intestine, brain, testis and
ovarian.
Fig. 6. Hepatopancreas and gill tissues collected from crabs injected with V. anguillarum were compared with respect to MIF mRNA expression (relative to b-actin) using
Students t-tests. Bars represent mean S.E. (n ¼ 6). Statistical significance is indicated
with an asterisk (P < 0.05).
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W.-W. Li et al. / Fish & Shellfish Immunology 30 (2011) 324e329
Analysis of secondary structure suggested that Es-MIF protein
possesses four a-helices and five b-sheets, in the following order:
b1a1b2b3b4a2b5a3a4, which is almost similar to human MIF,
whose three-dimensional structure is comprised of two a-helices
and six b-sheets in the following order: b1a1b2b3b4a2b5b6 [25].
The little difference is the structure of carbon terminal, which in
human MIF has a b-sheet (b6) and Es-MIF has two small a-helices
(a3a4), which was found in H. diversicolor supertexta too [40]. So
the putative molecular modeling of Es-MIF also has similar structure to that of human MIF. On account of the analysis above, we can
assume that Es-MIF protein may have similar biological functions to
human MIF.
Information extracted from Es-MIF tissue-dependent mRNA
expression may offer useful cues when speculating function. EsMIF transcripts were detected in all tissues examined, including
hepatopancreas, hemocytes, gills, brain, muscle, heart, intestine,
stomach, testis and ovarian. In mammals, MIF is ubiquitously
expressed in various cells and tissues such as the lung, the skin,
gastrointestinal and several tissues of the endocrine system
[7, 41-43], and our study was consistent with the ubiquitous
expression reported before. Notably, Es-MIF mRNA expression was
highest in hepatopancreas, and the possibility exists that Es-MIF’s
wide distribution among tissue types may be the result of hemocyte infiltration. Further, the high level of Es-MIF mRNA observed in
hepatopancreas may imply the hepatopancreas’ participation in
immune response, as does its expression in gill, which undergoes
constant bacterial challenges due to direct water exposure.
Current knowledge on acute response of MIF is mostly limited to
cultured cells (in vitro systems). Macrophage MIF can be released
after stimulation with microbial products that include bacterial
endotoxin (LPS), exotoxins, streptococcal pyrogenic exotoxin A,
malaria pigment, gram-negative and gram-positive bacteria,
mycobacteria, and proinflammatory cytokines [3,12,44], The doseeresponse curves after most of these stimulating were usually
bell-shaped. Moreover, time course of MIF release depends on cell
type, culture conditions and stimulating cytokine [45]. In this study,
we investigated the responsiveness of Es-MIF expression to an
immune challenge in order to elucidate potential involvement in
the invertebrate innate immune system. We observed a timedependent upregulation in Es-MIF expression following V. anguillarum stimulation, with a significant increase observed at 4 h postinjection (an 5.9-fold increase relative to the vehicle control), and
we observed a total of four expression peaks in response to a single
injection, after 4 h, 6 h (9.0-fold), 8 h (16.5-fold) and 12 h (4.6-fold).
The expression of the Es-MIF after immune challenge was determined in both hepatopancreas and gill tissues in E. sinensis. Es-MIF
mRNA levels in hepatopancreas increased sharply 6 h after exposure to V. anguillarum, and decreased down after 16 h post-exposure. In contrast, Es-MIF mRNA levels in gill were only slightly
increased after 8 and 12 h post-challenge. Control reactions, in
which cells were not induced with V. anguillarum, yielded no
significant increase in expression levels. The results showed that
Es-MIF was stimuli induced, and biologically relevant to crab
immune or inflammatory response to bacterial and may also play
a central role in the inflammatory response to bacterial.
To data, few reports pay attention to the acute response of MIF in
in vivo systems of aquatic animals, only available in vivo study was
performed in fish T. nigroviridis, TnMIF mRNA levels in spleen
increased sharply 3 h after exposure to LPS and decreased 12 h
post-exposure, and TnMIF mRNA levels in head kidney were only
slightly increased 3 and 24 h post-challenge [37]. In the study of
H. diversicolor supertexta [40], MIF expression level at 24, 48 h after
Vibrio parahaemolyticus injection was up-regulated significantly.
Furthermore, MIF is rapidly induced by some proinflammatory
effector molecules and pathogen components [7,45,46]. Recently,
emerging evidence demonstrated that MIF has a central role as
a regulator of innate immune and inflammatory responses, and the
implications it might have for the development of new therapies for
human autoimmune diseases, sepsis and other inflammatory
diseases [47,48]. Considering that MIF is constitutively expressed in
many cells [23,49] and exhibits catalytic properties reminiscent of
certain cellular enzymes [50,51], we reasoned that it was possible
that MIF interacts with intracellular proteins. JAB1 (Jun activating
binding protein 1) was initially discovered as a coactivator of AP-1
transcriptional activity [52], Jab1-mediated rescue of fibroblasts
from growth arrest is blocked by MIF. Amino acids 50e65 and Cys
60 of MIF are important for Jab1 binding and modulation. MIF may
act broadly to negatively regulate Jab1-controlled pathways and
that the MIF-Jab1 interaction may provide a molecular basis for key
activities of MIF [53], which need to be further studied in E. sinensis.
Our findings buttress those previously reported in other aquatic
animals suggest that Es-MIF expression is immuno responsive, and
Es-MIF may possess immune functionality in E. sinensis. However,
its mechanism of action is incompletely understood. So, the role of
MIF proteins or their therapeutic manipulation in immune diseases
is largely unexplored worthy territory, and the analysis of Es-MIF
action in immune responses could also yield valuable insights.
In conclusion, we have cloned the Es-MIF cDNA from Chinese
mitten crab E. sinensis which may help us to understand the origin
and evolution of the immune system as well as structure and
function of the immune system in crab. The expression of Es-MIF
was up-regulated following bacterial infection but was not changed
after PBS exposure which suggests that the Es-MIF gene may play
a role in the crab immune response.
Acknowledgments
This research was supported by the National Natural Science
Foundation of China (No. 30671607, 30972241).
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