Engineering, 2013, 5, 220-225
http://dx.doi.org/10.4236/eng.2013.510B046 Published Online October 2013 (http://www.scirp.org/journal/eng)
Copyright © 2013 SciRes. ENG
Molecular Cloning, Characterization and Expression
Analysis of Macrohage-Colony Stimulating Factor 2 Gene
from Grass Carp (Ctenopharyngodon idellus)
Linyong Du, Lei Qin, Shangnian Wang, Lu Yang, Kun Yang, Xinyan Wang, Hong Zho u
School of Life Science and Technology, University of Electronic Science and Technology of China,
Chengdu, China
Email: zhouhongzh@uestc.edu.cn
Received June 2013
ABSTRACT
Macrophage colony-stimulating factor (CSF-1/M-CSF) is a key factor for the differentiation, growth and survival of
monocytes/macrophages and osteoclasts. The functions of M-CSF have been well characterized in mammals. In this
study, we have cloned and sequenced the cDNA of M-CSF2 in grass carp. The grass carp M-CSF2 cDNA was 1487 bp
in length, containing an open read ing fr ame of 855 bp that encodes 284 amino acids. The deduced protein of grass carp
M-CSF2 possessed s ame domains similar to its mammalian counterparts. Multiple alignments and phylogenetic tree
indicate that the grass carp M-CSF2 exhibits close evolutionary relationship with its counterparts in other teleosts.
Lastly, the tissue distr ibution results also showed that the grass carp M-CSF2 transcript was dominantly expressed in
head kidney, kidney and spleen in vivo.
Keywords: Grass Carp; M-CSF2; Macrophages; Cloning; Sequence Analysis; Tissue Distribution
1. Introduction
From an evolutionary standpoint, the macrophages and
their functions are quite conser ved in several species, and
these cells have been identified in almos t all multicellular
organisms [1,2]. Macrophage activation is thought to
play a key role in innate immune response. Moreover,
macrophages also can secrete several cytokines and
chemokines to regulate adaptive immune response [3,4].
M-CSF (M-CSF1 in teleosts) is an important growth and
differentiation factor of macrophages in both of fish and
mammals [1,2,5]. M-CSF has also been demonstrated to
be involved in several biological processes, such as bone
metabolism, inflammation and pregnancy in mammals
[6-8]. All fo r ms of M-CSF initiate their effects mediated
by its receptor MCSFR, a memb er of the class III recep-
tor tyrosine kinase famil y which encoded by the c-fms
protooncogen [9].
Mammalian MCSF has three isoforms, a secreted gly-
coprotein, a secreted proteoglycan and a cell surface
glycoprotein. These isoforms are genera ted via a lterna-
tive splicing, post-trans lational modifications and pro-
teolytic processing [10]. The full biological activity of
M-CSF requ ir es all isof or ms, because these isoforms
exert specific functions on target cells which express
MCSFR at their surface and they play distinct roles in
inflammation and immunity [11]. Recently, several fish
M-CSF genes have been cloned, however, the known
fish M-CSF genes do not generate mammalian-equiva-
lent splicing variants [1,2]. In addition, two paralogous
M-CSF which may be generated by whole genome/
chromosome duplication event are identified in several
teleosts [2]. In spite of these, several evidences showed
the presence of functional homologous of M-CSF in sev-
eral low er vertebrates. The CSF-1 d oma in and the im-
portant cysteine residues required for formation of the
M-CSF in mammalian and teleost M-CSF are conserved
[2]. Furthermore, the recombinant fish M-CSFs have
been found to be biologically active like their homologs
in mammals, inducing differentiation and proliferation of
fish monocytes and influencing pro-inflammatory gene
expression in macrophages [1,12].
The grass carp (Ctenopharyngodon idellus) is a com-
mercially important freshwater fish and popularly cul-
tured in China and other Asian countries. In recent years,
with the rapid development of the commercial aquacul-
ture of grass carp in China, owing to the high density
breeding that causes deleterious changes in the environ-
ment and the increase of susceptibilities of fish to infec-
tions, grass carp has been exposed to serious diseases,
such as hemorrhagic disease, rotted gill disease and red
skin dise ase, causing heavy economic loss es. Sinc e
M-CSF represents one of the most important mediators
L. Y. DU ET AL.
Copyright © 2013 SciRes. ENG
221
of innate immune response to eliminate microorganism
infections, the studies on grass carp M-CSF would give a
valuable insight into the control of diseases in aquacul-
ture industry. In our study, we iso lated grass carp M-
CSF2 cDNA using the homology cloning approach, per-
formed sequence analysis and phylogeny analysis, and
revealed its expression patterns in various organs.
2. Materials and Methods
2.1. Animals
One-year-old Chinese grass carp, weighting 0.5 - 0.75 kg,
were purchased fro m Chengdu Tongwei Aquatic Science
and Technology Company (Chengdu, China). Tissues for
expression analysis were obtained from the freshly killed
fish according to the Regulation of Animal Use in Si-
chuan province, China.
2.2. Total RNA Extraction and cDNA Synthesis
Total RNA in the organs for expression analysis was
extracted by using Tripure (Roche, USA) according to
the manufacturer’s instructions. Five μg of total RNA
was subjected to reverse transcription by using Oligo
(dT)18 as the prime r with M-MLV reverse transcriptase
(Promega, Madison, MI) and s tored the cDNA at 80˚C.
2.3. Cloning of Grass Carp M-CSF2 cDNA
Partial sequences of grass carp M-CSF2 was identified
by homology-based PCR using other known teleost
M-CSF2 sequences as references, the degenerated pri-
mers were shown in Table 1. The PCR fragments were
sequenced. Based on the obtained fragment, RACE was
performed with gene specific primers (Table 1) follow-
ing the manufacturer’s instructions of GeneRacer Kit
(Life Technologies, Shanghai, China). The PCR frag-
ments was s equenced and assembled. Finally, we vali-
dated the full-length coding sequences (CDSs) of grass
carp M-CSF2 with Pfu DNA polymerase (Promega).
2.4. Sequence Analysis and Phylogeny Analysis
The open reading frames and deduced protein sequences
of grass carp M-CSF2 was identified by ORF finder [13].
The signal peptide was predicted using the SignalP 4.1
program and the transmembrane domain was predicted
using the TMpred program [14,15]. Characteristic do-
mains of the deduced proteins were predicted by SMART
program [16]. The identity analysis by using nucleotide
and protein BLAST programs
(www.ncbi.nlm.nih.gov/BLAST/). Mul tip le alignments
between the deduced amino acid sequence of grass carp
M-CSF2 and those of other known species M-CSF was
conducted with DNAMAN software (Lynnon Biosoft,
Table 1. Primers used for gene cloning and expression
analysis.
Na me Sequence (5’to 3’) Objects
M-CSF2 F CCAAGGGGAAGAATGTGTGTG cloning
M-CSF2 R GGGAGTAGATGTTGAGGATGAG
M-CSF2 5N1 CCATGCTCAGGTGTTTTCTCTC 5’-RACE
M-CSF2 5N2 CATCATGGAAAGGGGAACACACAG
M-CSF2 3N1 GAGAGAAAACACCTGAGCATGG 3’-RACE
M-CSF2 3N2 GCTTGAACTGTTTAACGTCCACTTC
M-CSF2
Realtime F CCTTGCAAACATGCCATAACCAT MCSF2
expression
M-CSF2
Realtime R CCATGCTCAGGTGTTTTCTCT
B-actin QF AGCCATCCTTCTTGGGTATG β-actin
expression
B-actin QR GGTGGGGCGATGATCTTGAT
Quebec, Canada). Phylogenetic tree was constructed by
using MEGA 4 (www.megasoftware.net) based on Neigh-
bor-Joining method with the bootstrapping of 1000 repe-
titions [17]. The 3D structure model of CSF-1 domain of
grass carp M-CSF2 was performed using the SWISS-
MODEL program (http:// swissmodel.expasy.org).
2.5. Expression Patterns of M-CSF in Vivo
Total RNA was extracted from the thymus, head kidney,
liver, spleen, gill, intestine and muscle , the cDNAs were
prepared by reverse transcription as described previously.
The gene-specific primers for grass carp M-CSF2 were
designed (Table 1) and used for detecting its transcript in
different tissues by real-time quantitative PCR (qPCR).
In our study, qPCR was performed on the Bio-Rad
CFX96 Real-time detection system (Bio-Rad Laborato-
ries, Inc., Hercules, CA) in a final volume of 20 μl with
10 μl iQTMSYBRTM Green Su pe rmi x (Bio-Rad Labora
tories, Inc., Her cules, CA) following the procedures in
our previous study. β-actin was used as a internal control.
Data were analyzed using the CFX manager and nor ma-
lized to β-actin after correcting fo r differences in amp li-
fication efficiency.
3. Results
3.1. Molecular Cloning Grass Carp M-CSF2
cDNA
The complete cDNA and deduced amin o acid sequences
of grass carp M-CSF2 (Genebank ID: KF113858) are
shown in Figure 1. The grass carp M-CSF2 cDNA se-
quence is 1487 bp in length with a 216 bp 5’-untran-
slated region (UT R ), a 416 bp 3’-UTR with the latter
containing a cytokine RNA instability moti f (ATTTA)
and a polyadenylation signal (AATAAA) nucleotide up-
L. Y. DU ET AL.
Copyright © 2013 SciRes. ENG
222
Figure 1. The cDNA sequences and deduced amino acids of
grass carp M-CFS2. The translation start codon and stop
codon are boxed. The signal peptide is underlined. The po-
tential N-glycosylation sites are in bold. Within 3’UTR the
putative ATTTA instability motifs are underlined and in
bold.
stream of the poly (A) tail. Grass carp M-CSF2 has an
855 bp open reading f ra me (ORF) encoding a 284-ami no
acid polypeptide.
The putative protein of M-CSF2 contained one N-
glycosylation sites, and its predicted molecular weight is
32.2 kDa and theoretical isoelectric point is 6.17 (data
not shown). The putative protein of grass carp M-CSF2
showed 88.89% and 53.31% identities to its homologs in
zebrafish and rainbow trout, respectively (Table 2).
When compared with M-CSFs in other species, grass
carp M-CSF2 shared 23.51% - 50.63% identity with
them (Table 2). In addition, alignment of protein se-
quence of grass carp M-CSF2 with other species M-CSF
revealed that they harbored an area of strong amin o acid
conservation encoding the CFS-1 d oma in (Figure 2, 4A).
Phylogenetic tree revealed that grass carp M-CSF2 was
close to the counterparts in fish and far from those of the
mammalian (Figure 3). Despite the difference in size of
the translated proteins and lower homology between fish
and mammalian M-CSF, analysis of M-CSF using the
SMART program indicated that it has a similar modular
structure with other species M-CSF, containing a signal
peptide, a CSF-1 domain, a tra ns memb r ane do main and
an intracellular region (Figure 4(a)). To further under-
stand the spatial structure of teleost M-CSF2, the 3D
homology modeling of the grass carp M-CSF was per-
formed by using the human M-CSF (PDB ID: 1h mc) as a
template. Figure 4(b) showed that grass carp M-CSF
consisted 2 parallel β-shells and 5 a-helixes that formed a
global fold, which was similar to the structure of the oth-
er mammal ian M-CSF.
3.2. Tissue Distribution of Grass Ca rp M-CSF2
The constitutive expression of grass carp M-CSF2 was
detected in all of selected tissues. As shown in Figure 5,
M-CSF mRNA was detected at the highest level in
spleen, at lesser extent in kidney, head kidney and thy-
mus , while low levels in liver, intestine and gill. Similar
basal expression pattern was observed in other fishes [2].
4. Discussion
M-CSF is a main factor for macrophage differentiation
and survival. Monocyte/Marophage is a major subset of
phagocytes in vertebrates which plays a vital role in in-
nate immune response [18]. The innate immune system is
supreme important for fish in defending infections and
maintaining homeostasis of the host immune system. In
order to better understand teleost innate immune system
and macrophage, we described the identification of grass
carp M-CSF2 molecule for th e first time. In addition, the
M-CSF2 functional domain and 3D structural model was
Tabel 2. CDS/amino acid identity of grass carp M-CSF2 with its homologs in other vertebrates (upper triangle = anino acid
identity; lower tria n g l e = CDS identity).
DNA/AA
1 2 3 4 5 6 7
1. Grass carp M-CSF2
27.64 88.89 23.51 53.31 50.63 39.29
2. Zebrafish M-CSF1
18.27 27.66 47.06 28.28 25.41 19.74
3. Zebrafish M-CSF2
80.79 22.16 23.42 53.25 51.20 39.95
4. Rainbow trout M-CSF1
16.09 32.72 16.47 25.68 23.67 18.67
5. Rainbow trout M-CSF2
37.94 20.79 41.84 17.56 62.56 42.25
6. Takifugu M-CSF2
39.74 19.21 41.78 18.23 55.56 41.95
7. Human M-CSF
19.21 10.15 21.88 9.02 18.02 19.05
L. Y. DU ET AL.
Copyright © 2013 SciRes. ENG
223
Figure 2. Multiple alignment of the predicted grass carp M-CSF2 deduced protein sequences with MCSFs in other species. The
conserved amino acid residues were highlighted by shadows. GcM-CSF2, grass carp M-CSF2; Zf M-CSF2, zebr afis h M-CSF2;
Rt M-CSF2, rainbow trout M-CSF2; Tf M-CSF2, takifugu M-CSF2; Hm M-CSF, human M-CSF.
Figure 3. Phylogenetic tree of M-CSF amino acid sequences
was constructed with the neighbor-joining algorithm by
using MEGA4 program. The branches were validated by
bootstrap analysis from 1000 replications, which are re-
presented by percentage in branch nodes. The GeneBank
accession numbers used in this study, zebrafish M-CSF2,
AM901599; zebrafish M-CSF1, AM901598; rainbow trout
M-CSF2, AM949839; rainbow trout M-CSF1, AJ555867;
goldfish M-CSF1, AM982798; takifuguM-CSF2, BAM75189;
mouse M-CSF, P07141; human M-CSF, P09603; Cow
M-CSF, O77709.
analyze d using bioinform a tics.
The grass carp M-CSF2 ORF was 855bp and showed a
relatively high degree of nucleotide sequence identity
with the three known fish M-CSF2 cDNAs, whereas,
when compared with fish M-CSF1 and mamma li an
M-CSF, it exhibited a low identities with those genes. In
3’-UTR of grass carp M-CSF2, one cytokine RNA in-
stability motif (ATTTA), which are typical cytokine
genes, and a p olyadenylation signal (AATAAA) were
found, indicating that the M-CSF2 mR NA expression
was tightly regulated [19,20].
The putative protein of grass carp M-CSF2 shared
similar molecular weight with other fish homologs. De-
spite that grass carp M-CSF exhibited low identity with
human M-CSF, both fish M-CSFs and human M-CSF
share similar structure: a signal peptide, a CSF-1 domain,
a transmembrane domain and an intracellular domain.
Furthermore, grass carp M-CSF2 share relatively higher
homology with human M-CSF at CSF-1 domain, which
is important for M-CSF function in mammals [2]. In ad-
dition, the alignment result of M-CS F s r ev ea led th a t
grass carp M-CSF2 are highly conserved with zebrafish
homologs. Meanwhile, zebrafish M-CSF2 gene is con-
served in syntenic chromosomal relationships compared
with huma n M-CSF [2]. Moreover, high similarity of 3D
models between human M-CSF and grass carp M-CSF2
was observed. All evidence described above indicates
47Gc M-CSF2 47Zf M-SCF2 47Rt M-CSF2 50Tf M-CSF2 50Hm M-CSF
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284Gc M-CSF2 284Zf M-SCF2 276Rt M-CSF2 271Tf M-CSF2 256Hm M-CSF
Consensus
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L. Y. DU ET AL.
Copyright © 2013 SciRes. ENG
224
(a)
(b)
Figure 4. (a) Domains of M-CSFs molecule. All M-CSFs
have signal peptide, CSF-1 domain, transmembrane do-
main and intracellular region; (b) 3D structure of CSF-1
domain in grass carp M-CSF2. Backbone ribbon and the
secondary structure topology are shown: blue arrows re-
present beta strands, red cylinders represent alpha helixes.
Figure 5. Expression pattern of grass carp M-CSF2 in dif-
ferent tissues. qPCRs were performed using primers speci-
c for IL-1β and β-actin. β-actin was amplified as internal
control, and then expressed as the ratio of the mRNA level
In. In, intestine; Gi, gill; T, thymus; Ms, musle; K, kidney;
L, liver; HK, head ki dney; Sp, spleen.
that the new ly cloned grass carp M-CSF2 is indeed the
ortholog of mammal M-CSF in gras s carp.
In mammals, M-CS F is known as an impor tant in-
flammato ry cytokine, in agreement with immunological
function in mammals, grass carp M-CSF2 transcript was
dominantly expressed in head kidney, spleen , kidney and
thymus. Actually, all of these tissues are considered as
the major lymphoid organs in teleosts.
In summa ry , our data showed that grass carp M-CSF2
was structurally similar to its counterpart in mammals
and exhibited relatively higher expression level in im-
mune-related tissues, providing the basis for further in-
vestigation of its immu n e role in grass carp.
5. Acknowledgements
This work was supported by grant from the National
Natural Science Foundation of China (31101877).
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