Tài liệu Báo cáo khoa học: Cloning, characterization and expression analysis of interleukin-10 from

Cloning, characterization and expression analysis of interleukin-10

from the common carp, Cyprinus carpio L.

Ram Savan1

, Daisuke Igawa2 and Masahiro Sakai2

1

United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto, Japan; 2

Faculty of Agriculture,

Miyazaki University, Miyazaki, Japan

Interleukin (IL)-10 was cloned from the common carp

(Cyprinus carpio L.) using IL-10 primers from carp head

kidney following stimulation with concanavalin A and

lipopolysaccharide. The cDNA consisted of a 1096 bp se￾quence containing a 55 bp 5¢ untranslated region and a

498 bp 3¢ untranslated region. An open reading frame of

543 bp encoded a putative 180 amino acid protein with a

putative signal peptide of 22 amino acids. The signature

motif of IL-10 is conserved in carp sequence. A 2083 bp

genomic sequence of carp IL-10 was found to contain five

exons interrupted by four introns. With the exception of

much more compact introns, the genomic structure was

similar to that of mammalian IL-10. By homology, phylo￾geny and genomic analyses, the carp gene cloned was des￾ignated as IL-10. Carp IL-10 was expressed in head, kidney,

liver, spleen and intestine during the resting phase. The gene

was also expressed in head kidney and liver following in vitro

stimulation with lipopolysaccharide.

Keywords: cytokines; interleukin; innate immunity; fish;

expression analysis.

Cytokines play a significant role in initiating and regulating

the inflammatory process, which is an important defense

system in innate immunity. Cytokines are subdivided into

families such as interleukins (ILs), lymphokines, growth

factors, interferons (IFNs) and chemokines. IL-10, initially

known as cytokine synthesis inhibitory factor, is a multi￾functional cytokine and demonstrates immunosuppressive

function. The main function of IL-10 seems to be regulation

of immunity and the inflammatory response, thereby

minimizing damage to the host induced by response to a

pathogen or by the self-immune system. IL-10 inhibits the

activation of macrophages/monocytes, thereby inhibiting

cytokine synthesis, nitric oxide (NO) production and the

expression of other costimulatory molecules. Apart from

IL-10 [1], a host of IL-10 family members such as IL-19 [2],

IL-20 [3], IL-22 [1], IL-24 [4] and IL-26 [5], have been

reported. Several IL-10 viral homologues have also been

reported [6], which mimic the activities of IL-10, suppressing

the immune system of the host to facilitate its survival [7].

As innate immunity is known to be important in the

defense of pathogens, isolation and characterization of

cytokines is of prime importance. Only a few cytokines and

chemokines are known in fish, where they have been cloned

either by expressed sequence tag (EST) analysis or by PCR￾mediated homology cloning. Among the cytokines, CC [8,9]

and CXC [10] chemokines, IL-1b [11], tumor necrosis

factor-a [12], transforming growth factor [13,14], IL-8 [15]

and IFN [16], have all been cloned in fish.

Recently, IL-10 homologues from torafugu (Taki￾fugu rubripes) and spotted green puffer fish (Tetraodon

nigroviridis) have been submitted to the EMBL database

(accession numbers CAD62446 and CAD67773), facilita￾ted by the fugu sequencing project [17]. However, expres￾sion of IL-10 has not been reported in fish. This is the first

report of an investigation of the expression patterns of IL￾10 in fish, in different tissues and its inducibility, when

stimulated with lipopolysaccharide (LPS). The presence of

IL-10 in fish gives significant insight on the regulation of

the immune response in fish. By homology, phylogeny and

genome analyses, the carp gene cloned was confirmed as

IL-10.

Materials and methods

Fish

Common carp (mean weight 100 g) was obtained from

Sunaso fisheries farm (Miyazaki, Japan). The fish were

acclimatized in an aerated fresh water tank at 20 C, under

a natural photoperiod, and fed for 2 weeks, prior to use in

the study.

Cloning and characterization of the carp IL-10 gene

A carp cDNA library, produced following stimulation with

concanavalin A and LPS [18], was used to isolate the IL-10

gene, employing IL-10-Fw2 and IL-10-Rv2 primers

(Table 1), which were designed based on the conserved

regions of puffer fish and mammalian IL-10. PCR was

performed using a PTC-200 (MJ Research, Waltham, MA,

USA) with 30 reaction cycles of: 30 s at 94 C, 30 s at 58 C

Correspondence to M. Sakai, Faculty of Agriculture, Miyazaki

University, Gakuen kibanadai nishi 1-1, Miyazaki 889-2192, Japan.

Fax: + 81 985 587219, Tel.: + 81 985 587219,

E-mail: [email protected]

Abbreviations: EST, expressed sequence tag; IFN, interferon;

IL, interleukin; LPS, lipopolysaccharide; NO, nitric oxide;

UTR, untranslated region.

(Received 7 August 2003, revised 16 September 2003,

accepted 25 September 2003)

Eur. J. Biochem. 270, 4647–4654 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03854.x