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Molecular cloning and characterization of macrophage migration inhibitory
factor from small abalone Haliotis diversicolor supertexta
Baozhen Wanga, Ziping Zhangb,**, Yilei Wanga,*, Zhihua Zoua, Guodong Wanga, Shuhong Wanga,
Xiwei Jiaa, Peng Lina
aThe Key Laboratory of Science and Technology for Aquaculture and Food Safety, Fisheries College, Jimei University, Yindou Road #43, Xiamen, Fujian 361021, China
bDepartment of Chemistry & Biochemistry, Texas State University, San Marcos, TX 78666, USA
a r t i c l e i n f o
Received 7 January 2009
Received in revised form
15 April 2009
Accepted 23 April 2009
Available online 6 May 2009
Haliotis disversicolor supertexta
a b s t r a c t
The macrophage migration inhibitory factor (mif) cDNA and its genome were cloned from small abalone
Haliotis diversicolor supertexta. Small abalone mif (samif) was originally identified from an expressed
sequence tag (EST) fragment from a normalized cDNA library. It’s 50untranslated region (UTR) was
obtained by 50rapid amplification of cDNA end (RACE) techniques and its genomic DNA was cloned by
PCR. The full-length cDNA of samif was of 535 bp, consisting of a 50-terminal UTR of 49 bp, an open
reading frame of 384 bp and a 30-terminal UTR of 102 bp. The deduced protein was composed of 128
amino acids, with an estimated molecular mass of 14.0 kDa and a predicted pI of 6.90. The full-length
samif genomic DNA comprises 3238 bp, containing three exons and two introns. Real time quantitative
PCR analysis revealed that samif gene is constitutively expressed in 6 selected tissues, and its expression
level in hepatopancreas is higher than that in the other tissues (p < 0.01). Samif expression level in the
hepatopancreas at 24 and 48 h after Vibrio parahaemolyticus injection was upregulated significantly
(p < 0.01), but there was no significant change after exposure to tributyltin (TBT) (p > 0.05).
? 2009 Elsevier Ltd. All rights reserved.
Macrophage migration inhibitory factor (MIF) was first discov-
ered as a lymphokine derived from activated T cells that inhibited
the random migration of macrophages in 1966 [1,2]. Currently it is
considered to be a multifunctional protein that can manifest itself
as a cytokine, growth factor, hormone and enzyme [3,4]. Besides its
well-defined role as a unique cytokine and critical mediator in
acute and chronic inflammatory diseases, autoimmune diseases,
cell proliferation and tumor angiogenesis , MIF also plays other
important roles, which include developmental regulation of life
cycle , axis formation and neural development  and embry-
onic development . MIF is an ancient and conserved biomole-
cule, and may associate with the evolution of the immune system
[9,10]. Up to now, mif cDNAs have been reported in many species,
such as: nematode [11–13], arthropod , chordate , amphibian
, fish , and so on. Even in single-celled parasitic protozoa
[6,15], mif ortholog is identified. However, MIF has not been
reported in mollusca so far, except for a sequence which has been
deposited into GenBank without any analysis very recently.
Small abalone Haliotis diversicolor supertexta is the most
commercially important cultured abalone in southern China. Since
late 2000, outbreaks of mass mortality among cultured abalone have
caused catastrophic losses to aquaculturists. Vibriosis is one of the
majordiseases thatoccur in abalone and Vibrio parahaemolyticus has
previously been described to be a pathogen of cultured small
abalone [16–18]. Some biochemical immunity factors and genes
have been reported to respond to V. parahaemolyticus challenge in
this species [19,20]. In addition, there is growing awareness that
pollution may influence the increasing disease incidences reported
in marine animals, possibly by inducing immunosuppression that in
turn would severely compromise host defense against pathogens
. Tributyltin (TBT) is one organic pollutant known to interfere
with hormone metabolism, especially the androgenic effect leading
to male imposex in gastropods which can perturb the immune
system as demonstrated in several species . Therefore, under-
standing the mechanism of immune systems of small abalone in
response to bacterial infection and TBT exposure is essential for
managing disease outbreaks and may contribute to the sustained
development of abalone culture.
In this article, the mif full length cDNA and its genome were
cloned from small abalone H. diversicolor supertexta, and its
* Corresponding author. Tel.: þ86 592 618 2723; fax: þ86 592 618 1476.
** Corresponding author. Tel.: þ1 512 245 0358; fax: þ1 512 245 1922.
E-mail addresses: email@example.com (Z. Zhang), firstname.lastname@example.org
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Fish & Shellfish Immunology 27 (2009) 57–64
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responses to pathogen challenge and TBT exposure are also
described. These results may allow us to gain more insight into the
genesis and evolution of mif gene and immunological function in
2. Materials and methods
Adult small abalone, H. diversicolor supertexta (body length
4.50 ? 0.50 cm, weight 7.55 ? 2.10 g), were collected from a local
commercial farm (Futian, Dongshan, Fujian Province). Abalones
were maintained inpolyethylene tanks, each containing 20 animals
in 50 L aerated sand-filtered seawater at 23–25?C, and fed with
kelp. The culture medium was renewed with fresh seawater every
day. Abalones were left undisturbed for 2 weeks to acclimate to
their environment before bacterial challenge and TBT exposure.
2.2. Bacterial infection and TBT exposure
For each trial (bacterial infection, TBT exposure and controls),
three replicates were prepared holding 20 abalones per tank. In the
bacterial infection experiment, abalones were challenged by
injecting either 50 mL of V. parahaemolyticus (isolated from diseased
abalone, ) in 0.9% NaCl (6.7 ? l07cfu/mL), or 50 mL of 0.9% NaCl
(as control) into their pleopod muscle. After injection, the abalones
were returned to their original tanks containing seawater at the
same temperature. The hepatopancreas of control or challenged
abalone were collected at 8, 24, 48 and 96 h post-challenge.
Samples were collected and frozen at ?80?C until analysis.
In TBT exposure experiment, TBTCl (Sigma) was dissolved to 2g
(Sn)/L in 100% ethanol as stock solution. The stock solution was
then diluted in fresh seawater to a final concentration of 1.75 mg
(Sn)/L for exposure. In control, an equal volume of fresh seawater
containing 0.1% of 100% ethanol was used instead of TBT solutions.
Fresh seawater with specific TBT concentration was exchanged
every day. Hepatopancreases were taken from both challenged and
control abalones at 8, 24, 48, 96 and 192 h post-challenge and
frozen at ?80?C until use.
In addition, hepatopancreas, muscle, ovary, gill, epipodium and
mantle were also sampled from small abalone without bacterial
infection and frozen at ?80?C until use.
2.3. Normalized cDNA library construction and EST analysis
A normalized cDNA library was constructed from the liver of
small abalone, using the combination of SMART technique and DSN
treatment. Random sequencing of the library using M13 primer
yielded 2473 successful sequencing reactions. BLAST analysis of all
the 2473 EST sequences revealed that an EST of 440 bp (clone NO:
JXW-23H04.ab1) was highly similar to the identified mif from
Ascaris suum, Petromyzon marinus, Rattus norvegicus and Homo
sapiens, therefore, this EST sequence was selected for further
cloning of the full length cDNA of samif.
2.4. Cloning the 50untranslated region of samif cDNA
The selected EST clone was re-sequenced with the M13 sense
and antisense primers on a MegaBACE500 sequencer. The entire
open reading frame and 30untranslated region were obtained.
Based on the EST sequence, samif 50special primers (Table 1) were
designed for amplification of cDNA 50ends (RACE). The universal
primers used for 50-RACE were UPM and NUP. 50-RACE was carried
out by using an SMART? RACE cDNA amplification kit (Clontech,
USA) according to the manufacturer’s instructions. Nest PCR was
used for the amplification of 50UTR. The PCR programs werecarried
out at 94?C for 3 min, followed by 35 cycles of 94?C for 30 s, 68?C
for 30 s, 72?C for 2 min and a final extension step at 72?C for
10 min. The PCR products were resolved by electrophoresis on 1%
agarose gel. The fragments of interest were excised and then
purified byGel Extraction Kit (Generay Biotech, China). The purified
fragments were then cloned into pMD18-T vectors (Takara, Japan),
and propagated in Escherichia coli (JM109) competent cells. The
plasmids isolated from positive clones were sequenced.
2.5. Cloning the genomic DNA of samif
Genomic DNA was isolated from small abalone hepatopancreas
by using Tissue/Cell DNA Mini Kit (Watson, China). In order to
analyze the structure of samif gene, samif genomic DNA was
amplified by PCR. The primers, samif full sense and samif full
antisense, designed according to the full sequence of samif cDNA
and the knowledge of the positions of introns in the zebra fish MIF
genome, were used to amplify the genomic DNA with PCR
programs at 94?C for 3 min, 35 cycles (94?C 30 s, 55?C 30 s, 72?C
6 min), 72?C elongation for 10 min (Table 1). The fragments of
interest were excised and then purified by Gel Extraction Kit
(Generay Biotech, China). The purified fragments were then cloned
into pMD18-T vectors (Takara, Japan), propagated in E. coli (JM109)
competent cells. The positive clones were selected and sent to the
invitrogen company for sequencing. The primers MIF genome.w2f,
MIF genome.w1f(c) and MIF genome.w1f were used to sequence
the intermedial sequence of samif genomic DNA. All genomic DNA
fragments were then overlapped to obtain the full-length mif
genomic DNA of small abalone.
2.6. Bioinformatics analysis
at (http://cn.expasy.org/tools/pi_tool.html). The signal sequence
was identified using the program SignalP (http://www.cbs.dtu.dk/
services/SignalP). The SCRATCH Protein Predictor server (http://
secondary structures. Three-dimensional structure was predicted
using swiss-model at http://www.expasy.org/swissmod. Protein
multiple-alignments were performed with T-Coffee (http://www.
ch.embnet.org/index.html). Phylogenies of protein sequences were
estimated using MEGA 4.0 using neighbor-joining method.
2.7. Real time quantitative PCR
The analysis of samif tissue distribution and expression after
challenge were performed by real time quantitative PCR using SYBR
Green I. In general, primers for samif and beta-actin were designed
primer3_www.cgi) (Table 1) and tested to ensure amplification of
single discrete bands with no primer-dimers. An aliquot of 3 mg of
RNA pre-treated with DNase I were used as template for total cDNA
synthesis in 20 mL reactions with random hexamers using the M-
MLV RT Usage information (Promega). For real time PCR, an amount
of cDNA corresponding to 25 ng of input RNA was used in each
reaction. Reactions were performed with the SYBR Green PCR
Master Mix (Applied Biosystems), and analyzed in the ABI 7500 real
time System. The cycling conditions for both samif and b-actinwere
as follows: 1 min at 95?C, followed by 40 cycles (15 s at 95?C,1 min
at 60?C). Melting curves were also plotted (60–90?C) in order to
make sure that a single PCR product was amplified for each pair of
primers. The comparative threshold cycle (CT) method (user
Bulletin#2, the ABI PrismR 7700 Sequence detector (PE Applied
Biosystems)) was used to calculate the relative concentrations. This
B. Wang et al. / Fish & Shellfish Immunology 27 (2009) 57–64 58
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method involves obtaining CT values for the samif, normalizing to
the housekeepinggene,b-actin (GenBank accessionno. AAS00498),
and comparing the relative expression level. Experiments were
performed routinely with five challenged abalones and five control
abalones with values presented as 2?66CTfor the expression levels
of samif normalized with b-actin (6CT ¼ CT of samif minus CT of
beta-actin, 66CT ¼ 6CT of challenged sample minus 6CT of
calibrator sample). Data were expressed as mean and standard
error of the mean (SEM) unless otherwise stated. Five separate
individuals at each time were tested, each assayed in triplicate.
Statistical analysis of the normalized CT values was performed with
Student’s t-test using SPSS. Differences were considered significant
at p < 0.05 (two-tailed test).
3.1. cDNA cloning, characterization and homology analysis of samif
The full-length cDNA of samif was obtained by overlapping the
re-sequenced ESTclone and the cloned sequence of 50untranslated
region. The samif open reading frame is of 384 bp in length. The 50
UTR locates 49 bp upstream of the putative start codon (ATG). The
30UTR of 102 bp nucleotides ends at a poly(A) tail. A non-canonical
polyadenylation signal site (ATTAAA) is located 61 bp downstream
of the stop codon (TAA). The sequence of this cDNA has been
deposited in the GenBank database under accession number
samif ORF encodes 128 amino acids, with a calculated molecular
mass of 14.0 kDa and a theoretical pI of 6.90. Protein Signal-peptide
prediction showed that the saMIF lacks an amino-terminal signal
peptide, indicating that it is a non-secretary protein. The amino-
terminal proline residue (Fig.1, positions P2) which is crucial for the
catalytic activity of isomerase [6,7,9–15] is also conserved in samif.
Predictions of secondary structure show that there are five beta-
sheets and four alpha-helices (shown in Fig. 1) in samif, which is
similar to that of human mif.
To examine the saMIF relationship of small abalone to the
various species, a phylogenetic tree was constructed using the
neighbor-joining method with the amino acid sequences of small
abalone and the other known MIFs (Fig. 3). The phylogenetic tree
reveals a grouping of MIF proteins into two main branches, one
comprising animal proteins, and the other one containing plant
protein. In the animal branch, the branching pattern of the resul-
tant phylogenetic tree corresponded essentially with the evolu-
tionary relationships among the species. Small abalone and Haliotis
discus discus form a cluster separate from the other species.
Mammalian, avian, amphibian, jawed fish, jawless fish, chordate,
arthropod and nematode form a respective cluster. The deduced
small abalone MIFamino acid sequence shares high homology with
that from H. discus discus (72% identity) and the identity of MIF
between small abalone and the other species is almost 40%.
3.2. Genome cloning of samif
The full-length samif genomic DNA (Genbank accession No.
FJ195326) was obtained by overlapping five genomic DNA frag-
ments. The obtainment of samif genomic DNA checked the accuracy
of samif cDNA sequence. The full-length samif genomic DNA
comprised 3238 bp, containing three exons and two introns
conserved in all selected species (Table 2). The length of three exons
is of 108 bp,173 bp and 103 bp respectively (Table 2). The length of
the first intron is of 2403 bp and that of the second intron is of
455 bp (Table 2). In the samif genomic DNA, the splice donor and
acceptor sequence of the two introns is 50GT-AG30(Table 2).
3.3. Expression analysis of the samif gene
As shown in Fig. 4, samif was constitutively expressed in all 6
selected tissues from healthy small abalone. Real time quantitative
PCR analysis revealed that the tissue with the highest expression
level of samif is hepatopancreas (p < 0.01).
Based on hepatopancreas expressing more than the other
selected tissues and in order to better understanding the biological
role of samif, we evaluated mif expression levels in hepatopancreas
of small abalone challenged by bacterial infection and TBTexposure
at different time courses. Samif expression level at 24, 48 h after
(p < 0.01) (Fig. 5), while samif expression levels in hepatopancreas
post-TBT exposure were not significantly different compared to
controls (p > 0.05) (Fig. 6).
As we stated in introduction, MIF was found to be conserved
throughout species, from both invertebrates and vertebrates, even
Oligonucleotide primers used in this article.
MIF/EU28411450RACE out primer
50RACE inner primer
MIF full sense
MIF full antisense
MIF/EU284114 Real time quantitative
PCR sense primer
Real time quantitative
PCR antisense primer
beta-actin/AAS00498 Real time quantitative
PCR sense primer
Real time quantitative
PCR antisense primer
Fig. 1. The deduced amino acid sequences of small abalone MIF protein. The site of
catalytic activity is indicated as P. Beta-sheets are dropped shadow and alpha-helices
are underlined. The poly A signal sequences is bold.
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in single-celled parasitic protozoa. The presence of macrophage
migration inhibitory factor in mollusca from small abalone further
demonstrated that MIF is evolutionarily conserved. This conserva-
tion of MIF 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 . MIF was also found to be essential for axis
formation and neural development of Xenopus embryos .
Moreover, MIF may be useful as a marker of the life cycle, i.e.
Eimeria MIF was detectable in merozoite stage but not in sporozoite
stage . In species of Trichinella as well as the free-living nema-
tode C. elegans MIF is upregulated in adult worms, while consti-
tutively expressed inother stages[11,24].Basedonthe
Fig. 2. Multiple alignment of the MIF amino acid sequence between Haliotis diversicolor supertexta and other species. Species and gene names are abbreviated at the left and
represent, with GenBank Accession no. as follows: Haliotis diversicolor supertexta, ABX76741; Haliotis discus discus, ACJ65690; Homo sapiens, P14174; Mus musculus, P34884; Gallus
gallus, Q02960; Xenopus laevis, Q76BK2; Danio rerio, NP_001036786; Oncorhynchus mykiss, ABG54279; Myxine glutinosa, Q86BT2; Amblyomma americanum, Q9GUA9, Caenorhabditis
elegans, Q9U228; Leishmania major, CAJ06450. Sequence alignment was performed using the T-Coffee at http://www.ch.embnet.org/index.html. Consistency scores are shown by
color as:BAD AVG GOOD. The site of catalytic activity (P2), the site of isomerase activity (k33) and the sites of oxidoreductase activity (C57, C60and C81) are indicated with ;.
B. Wang et al. / Fish & Shellfish Immunology 27 (2009) 57–6460
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pathophysiologic and metabolic functions of MIF throughout
species, it is intelligible that MIF is very conserved in evolution.
The amino-terminal proline residue (Figs. 1 and 2, position P2)
which is crucial for the catalytic activity of isomerase [6,7,9–15] is
conserved in all selected species (shown in Fig. 2). In addition, the
invariant lysine residue (Fig. 2, position K33) observed in many
species, which contributes to the isomerase activity of the protein
, is also present in saMIF. Because crucial amino acid residues for
the isomerase activity of mammalian MIF were shared with saMIF,
we expected saMIF to have the same activity as mammalian MIF.
Mammalian MIF is distinguished by the presence of three
conserved cysteines (Fig. 2, Cys57, Cys60, and Cys81), the first two of
which define a CXXC motif that mediates thiol protein oxidore-
ductase activity [6,7,9–15]. Except that Cys57is conserved in saMIF,
the latter two cysteines as well as the CXXC motif, are absent in
saMIF. Therefore, whether small abalone has distinct oxidoreduc-
tase activities needs to be further studied.
Analysis of secondary structure suggested that samif possesses
four a-helices and five b-sheets,
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 .
The little difference is the structure of carbon terminal, which in
human MIF has a b-sheet (b6) and samif has two small a-helices
(a3a4). So the putative molecular modeling of saMIF also has
similar structure to that of human MIF (Fig. 7). On account of the
analysis above, we can assume that samif may have similar
biological functions to human MIF.
The genomic organization of samif gene shows a striking
similarity with that of H. sapiens, Mus musculus, R. norvegicus, Danio
rerio, Strongylocentrotus purpuratus and Caenorhabditis elegans,
which all include three exons and two introns (Table 2). The lengths
of the first exon and the second exon are the same as those of H.
sapiens, M. musculus, R. norvegicus, D. rerio and S. purpuratus, but
shorter than those of C. elegans. The length of the third exon is
different from all other selected species except S. purpuratus, longer
than that of human, mouse, rat, zebra fish, and nematode, which is
why saMIF (128 aa) is not similar to most known counterparts (115
aa) in length (shown in Fig. 2). The pattern characterized by 5’GT-
AG3’ is conserved in all selected species for spliceosomal introns.
All results demonstrate that the structure and organization of MIF
genes are highly conserved between small abalone and other
vertebrates during evolution.
Consistent with the available reports showing that mif is ubiq-
uitously expressed in both immune and non-immune cells
including various tissues [5,26,27], in this study, real time quanti-
tative PCR analysis revealed that samif gene is also constitutively
expressed in 6 selected tissues from healthy small abalone and its
expression level in hepatopancreas is higher than that in muscle,
Fig. 3. Phylogenetic tree of the MIF amino acid sequence between Haliotis diversicolor supertexta and other species. The abbreviations used for species are represent, with GenBank
Accession no. as follows: Homo sapiens, P14174; Macaca mulatta, Q6DN04; Mus musculus, P34884; Rattus norvegicus, P30904; Gallus gallus, Q02960; Xenopus laevis, Q76BK2; Danio
rerio, NP_001036786; Ictalurus punctatus, ABG54274; Fugu rubripes, AAW50794; Oncorhynchus mykiss, ABG54279; Salmo salar, ABG54277; Myxine glutinosa, Q86BT2; Haliotis
diversicolor supertexta, ABX76741; Haliotis discus discus, ACJ65690; Branchiostoma belcheri, Q698K1; Amblyomma americanum, Q9GUA9; Caenorhabditis elegans, Q9U228; Wuchereria
bancrofti, O44786; Trichuris trichiura, Q9U920; Arabidopsis thaliana, AAO50451. The number near node represents bootstrap values.
Ratio of samif/beta–actin
Fig. 4. Tissue distribution of samif in 6 selected tissues and organs with beta-actin as
endogenous control gene. Data are presented as means ? SEM of four separate indi-
viduals, each assayed in triplicate. Significant differences from controls were indicated:
**p < 0.01. H: hepatopancreases; M: Muscle; O: ovary; G: gill; T: mantle; E: Epipodium.
B. Wang et al. / Fish & Shellfish Immunology 27 (2009) 57–6461
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ovary, gill, epipodium and mantle (p < 0.01). However, gill, gonad,
heart and brain demonstratedhigherlevels of expression than liver,
intestine, skin, muscle, spleen and head kidney in Tetraodon
nigroviridis MIF (TnMIF) . One reason for the difference of
expression pattern may be the difference in immune systems.
Compared with fish, molluscs lack a consummate immune organ
and the hepatopancreas is its major organ of detoxification, while
mucosal immune systems (including gill) are the first line of
immune defense in fish. In addition, MIF was found to be essential
for axis formation and neural development of Xenopus embryos 
and is involved in the life cycle of some species [6,15]. We suggest
that small abalone and pufferfish may be at different gonad
development stages, which accounts for the difference of expres-
sion pattern. Further investigation is necessary to confirm the
direct or indirect involvement of mif in gonad development and to
explain the molecular mechanism.
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) and exotoxins (toxic shock syndrome toxin [TSST]
and streptococcal pyrogenic exotoxin A [SPEA]), malaria pigment,
Gram-negative and Gram-positive bacteria, mycobacteria, and
proinflammatory cytokines, such as tumor necrosis factor (TNF)-
a and interferon-g [28–30]. The dose–response curves after most of
these stimuli were usually bell-shaped. Moreover, time course of
MIF release depends on cell type, culture conditions and stimu-
lating cytokine . Few reports pay attention to the acute
response of MIF in in vivo systems of aquatic animals. Only one
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 .
However, the response of aquatic animals to whole pathogenic
organisms in an invivosystem has notbeen reportedyet. This study
is the first to use an in vivo system to study mif response to whole
pathogen infection in an aquatic animal. Samif in hepatopancreas
Ratio of samif/beta–actin
Fig. 5. The change of samif after V. parahaemolyticus challenge with beta-actin as
endogenous control gene. Data are presented as means ? SEM of five separate indi-
viduals, each assayed in triplicate. Significant differences from controls were indicated:
**p < 0.01.
Ratio of samif/beta–actin
1.75µ g/L TBT
Fig. 6. The change of samif after TBT exposure with beta-actin as endogenous control
gene. Data are presented as means ? SEM of five separate individuals, each assayed in
triplicate. There is no significant difference between treatment and control (p > 0.05).
Fig. 7. The three-dimensional ribbon structure compared the small abalone MIF model (A) with human MIF model (B) which was predicted by Swiss-model and Rasmol software.
The length of exons and introns of genomic MIF from different species.
NameGenBank no. RegionExon1 Exon2Exon3Intron1 Intron2
Homo sapiens MIF
Mus musculus MIF
Rattus norvegicus MIF
Danio rerio MIF
Strongylocentrotus purpuratus similar to MIF isoform 1
Haliotis diversicolor supertexta MIF
Caenorhabditis elegans MIF1
22 566 565–22 567 409
75 322 098–75 322 995
13 191 986–13 192 851
52 272 090–52 277 622
776(gt-ag)11 848 033–11 849 391
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was induced after V. parahaemolyticus infection and the upregula-
tion of samif after bacterial challenge suggests that MIF plays
a critical role in inflammatory response to infection and tissue
invasion. After V. parahaemolyticus infection, samif expression in
hepatopancreas increased significantly at 24 and 48 h (p < 0.01),
and then recovered to the original level after 96 h. However, the
expression sites and time course in abalone are different from fish.
Although molluscs do not have macrophage cells, emerging
evidences [32,33] are reported that gastropods may also have
macrophage-like cells, the identification of saMpeg1 published by
our group  and identification of samif in this study further
support this hypothesis. SaMPEG1 is a homolog of two mammalian
proteins that share homology with mammalian perforin, a cytolytic
and immune-regulatory protein of lymphocytes , which is an
effector molecule of a signal pathway. SaMpeg1 is highly expressed
in hepatopancreas at 96 h post-V. parahaemolyticus injection, while
samif expression in hepatopancreas increased significantly at 24
and 48 h (p < 0.01). We infer that samif may be a regulator in the
signal pathway of sampeg.
It is clear that the immune system is a target for TBT toxicity
[34,35]. However, no reports pay attention to the relationship
between TBT and genes related to immunity. Reports concerning
the immunotoxicity of TBT to molluscs are available, such as Mytilus
edulis , Tapes philippinarum  and Mya arenaria , suggest
that TBT decreases the number of haemocytes and inhibits their
phagocytosis. Our TBT exposure indicated that there was no
significant impact on expression of samif. SaMpeg1, another gene
we cloned, also did not change after TBT exposure . However,
we found the superoxide dismutase gene (Sod) is downregulated
after TBT exposure . SODs are a group of metalloenzymes that
catalyse the conversion of reactive superoxide anions (O2
hydrogen peroxide (H2O2), and are considered to play a role as
pivotal antioxidants . Considering all evidence above, we
suggest that TBT may have an impact on antioxidant enzymes and
their genes, but has little effect on genes related to immune func-
tion of haemocytes. TBT is known as an inhibitor of oxidative
phosphorylation, affecting the cell metabolism by stimulating the
production of adenosine triphosphate and by inhibiting its trans-
formation into adenosine diphosphate . Whether the concen-
tration of TBT is too low or high to activate mif needs to be further
In conclusion, we have cloned the MIF cDNA and its genome
from small abalone H. diversicolor supertexta 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 abalone. The
expression of samif was upregulated following bacterial infection
but was not changed after TBT exposure which suggests that the
samif gene may play a role in the immune response.
?) to yield
This work was supported by National High Technology Research
and Development program of China (863 program) project
(2002AA629220). Natural Science Foundation of China (20877034),
Science and Technology Foundation of Fujian Province (2007
N0048), the Committee of Xiamen Science and Technology, Xia-
men, Fujian, China (502Z20055024) and the Innovation Team
foundation of Jimei University (2008A001). We thank Miss Lynsdey
M. Kirk (Department of Chemistry and Biochemistry, Texas State
University, Texas, USA) for critical reading of the manuscript.
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