Gene structure of goose-type lysozyme in the mandarin fish Siniperca chuatsi with analysis on the lytic activity of its recombinant in Escherichia coli

Aquaculture 252 (2006) 106 – 113 www.elsevier.com/locate/aqua-online

Gene structure of goose-type lysozyme in the mandarin fish Siniperca chuatsi with analysis on the lytic activity of its recombinant in Escherichia coli B.J. Sun a,b , G.L. Wang a,b,c , H.X. Xie a,b , Q. Gao a,b , P. Nie a,b,⁎ a

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, Hubei Province, P.R. China b Laboratory of Fish Diseases, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, Hubei Province, P.R. China c College of Animal Science, Northwest Sci-Tech University of Agriculture & Forestry, Yangling 712100, Shanxi Province, P.R. China Received 20 January 2005; received in revised form 21 June 2005; accepted 25 July 2005

Abstract A goose-type lysozyme (g-lysozyme) gene has been cloned from the mandarin fish (Siniperca chuatsi), with its recombinant protein expressed in Escherichia coli. From the first transcription initiation site, the mandarin fish g-lysozyme gene extends 1307 nucleotides to the end of the 3′ untranslated region, and it contains 5 exons and 4 introns. The open reading frame of the glysozyme transcript has 582 nucleotides which encode a 194 amino acid peptide. The 5′ flanking region of mandarin fish glysozyme gene shows several common transcriptional factor binding sites when compared with that from Japanese flounder (Paralichthys olivaceus). The recombinant mandarin fish g-lysozyme was expressed in E. coli by using pET-32a vector, and the purified recombinant g-lysozyme shows lytic activity against Micrococcus lysodeikticus. © 2005 Elsevier B.V. All rights reserved. Keywords: g-type lysozyme; Gene organization; Promoter; Recombinant protein; Mandarin fish; Chinese perch; Siniperca chuatsi

1. Introduction Lysozyme is an enzyme which can hydrolyze 1, 4beta-linkages between N-acetyl-D-glucosamine and Nacetylmuramic acid in peptidoglycan heteropolymers of prokaryotic cell walls, leading to breakdown of bacteria Abbreviations: ORF, open reading frame; nt, nucleotides; aa, amino acid(s); UTR, untranslated region; RT-PCR, reverse transcription-PCR. ⁎ Corresponding author. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, Hubei Province, P.R. China. Tel.: +86 27 68780736; fax: +86 27 68780123. E-mail address: [email protected] (P. Nie). 0044-8486/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.07.046

(Jollés and Jollés, 1984). The research on lysozymes has long been an interest, and the first report on lysozymes appeared as early as in 1960s (Canfield and McMurry, 1967). Two different lysozymes have been found in vertebrates, i.e. chicken (c-) and goose (g-) types, which differ in molecular weight, amino acid composition and enzymatic properties (Prager and Jollès, 1996; Irwin and Gong, 2003). Goose-type lysozyme (g-lysozyme) was initially identified as an anti-bacterial enzyme in egg whites of several avian species, and in recent years genes of its homologues have been sequenced in mammals and fish (Irwin and Gong, 2003). In fish, the cDNA of c-type lysozyme has been reported from Japanese flounder Paralichthys olivaceus (Hikima et al.,

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2000), turbot Scophthalmus maximus, rainbow trout Oncorhynchus mykiss (Dautigny et al., 1991), zebrafish Danio rerio and common carp Cyprinus carpio, and the g-type lysozyme in Japanese flounder (Hikima et al., 2001), common carp and orange-spotted grouper Epinephelus coioides (Yin et al., 2003). But, the gene structure of g-lysozyme in fish has only been just discussed in Japanese flounder, and much more information is needed to understand the existence of such lysozyme in fish. In China, the mandarin fish or the so-called Chinese perch, Siniperca chuatsi (Basilewsky) (Perciformes) has a relatively high market value, and is widely cultured throughout the country with importance also in stocking fishery in lakes and reservoirs (Liu et al., 1998). However, outbreaks of diseases caused by parasites, bacteria and viruses have caused severe economic losses to the aquaculture industry, and in some cases the mortality can reach as high as 100% (He et al., 2002). Despite the economical importance of the fish and severe economic losses caused by diseases, little research has been carried out on the mandarin fish immune factors. The only reported literature has been related to genes of immunoglobulin (Ig) and a virus induced protein (Zhang et al., 2003; Sun and Nie, 2004). The present study was designed to identify the glysozyme gene of the mandarin fish, and the activity of its recombinant protein in E. coli. 2. Materials and methods 2.1. Cloning of g-type lysozyme cDNA by RACE-PCR Degenerate primers were designed from a conserved region obtained by comparing all known g-type lysozyme sequences, and all primers used in this paper are listed in Table 1. Two mandarin fish, weighing about 200 g each were injected with 400 μg LPS (Sigma,

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USA), respectively, and 3 days later the RNA was isolated using Trizol reagent (Invitrogen, USA) from the head kidney and reverse transcribed into cDNA by Powerscript II reverse transcriptase with CDS primer (SMART RACE cDNA Amplification kit, Clontech, USA). The PCR cycling conditions were 94 °C for 3 min followed by 30 cycles of 94 °C for 30 s, 54 °C for 30 s and 72 °C for 1 min, and then a final elongation step at 72 °C for 5 min. PCR products were cloned into pGEM-T vector (Promega, USA) and sequenced. To recover the full-length cDNA sequence, 3′ RACE and 5′ RACE were performed by using the gene specific primers and adaptor primers. The PCR cycling conditions were 94 °C for 3 min followed by 30 cycles of 94 °C for 30 s, 64 °C for 30 s and 72 °C for 1 min, and then a final elongation step at 72 °C for 5 min. The BLAST program from the National Center for Biotechnology Information was used to identify similar sequences. The multiple sequence alignments were performed using the CLUSTAL W 1.8 program, and the putative signal peptide was analyzed using the SignalIP software (Nielsen et al., 1997). 2.2. Cloning of g-type lysozyme genomic sequence and 5′ flanking region The 5′ flanking region was obtained using a genome walking approach, by constructing genomic libraries with a Universal Genome Walker™ kit (Clontech). The sequence of the 5′ flanking region was analyzed by TRANSFAC software for potential transcriptional factor binding sites (Wingender et al., 2000). 2.3. Production and purification of mandarin fish recombinant g-lysozyme in E. coli The PCR amplified g-type lysozyme gene fragment encoding the open reading frame was digested with

Table 1 Primers used for the mandarin fish g-lysozyme cloning and expression Name

Sequence (5′–3′)

Application

LF LR SclysF SclysR L51 L52 L31 L32 DNAF DNAD

GCATCACACA(CA)(CA)ATGGCA TTTGTACCACTGAGCTCTGGC GGAGGTACCATGGGTTATGGAAACATC CCAGATATCTTAAAAGCCTTCGTTG CCCTGGACTCTCTGGAGATGATGG TGATGGCAGCGATTAGAGCTGGA TCCTGGCTGGAGCACGGAGCAGC AGCAGCAGCTGAAAGGAGGGATAG CGTTGCTGCTCTCGCATTCCAG GGATTTGCTTTGCATTATGTTT

Conserved region cloning Conserved region cloning Expression in E. coli Expression in E. coli cDNA 5′ RACE, 1st round cDNA 5′ RACE, 2nd round cDNA 3′ RACE, 1st round and genomic walking cDNA 3′ RACE, 2nd round and genomic walking Genomic DNA cloning Genomic DNA cloning

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pellet was resuspended in ice-cold 1 × Ni–NTA binding buffer, lysed in 3 consecutive freezing (− 35 °C) and thaw (room temperature) cycles and sonicated on ice (20 s intervals, 10 min). The bacterial lysate was then centrifuged at 16,000 ×g for 30 min, and the supernatant was added to the Ni–NTA HisBind slurry and mixed gently by shaking for 1 h. After washing the column, the protein was eluted with 250 mM imidazole elution buffer. Purity of the recombinant protein was assessed on 12% SDS-PAGE gel, and the concentration was determined by Bradford method.

KpnI and HindIII, ligated into the pET-32a expression vector linearized with the same enzymes, and transformed into DH5α competent cells. After sequencing the positive clones to ensure in frame insertion, the pET32a-lysozyme construct was transformed into E. coli BL21 (DE3) strain for protein expression. The fusion protein was expressed by isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, and analyzed with SDS polyacrylamide gel (SDS-PAGE). Purification of recombinant proteins under native conditions was performed by using the Ni–NTA His·Bind Resins (Novagen). Briefly, the bacterial

CGTTGCTGCTCTCGCATTCCAGTTTCAAAGAGACCAGCAGAAGAGGAAACAGGAAAAATG M

60 1

GGTGGGTGCTTGTGTTATTTTACTGTTGATAGAACTTGACTTTCAGCTGTGAATGTGCTT

120

GGAAGAAAGAAACACTGGAATACTTATTCACAGGGAACAATAGTGTGTCTTATCACTTTT GTGTGGGTTATGTAACTGTAAAGTCCCTTGAGGTCCTCATGCTATGTGCTCATGTTGAAA ACTAATCTAATAACCTAAACATTTGCAGGTTATGGAAACATCATGAGGCTTGAAACTACT

180 240 300

G

Y

G

N

I

M

R

L

E

T

T

GGAGCTTCATGGGAAACAGCTCAGCAGGACAGTCTGGCATACTCAGGTAAGTAAACACTG G

A

S

W

E

T

A

Q

Q

D

S

L

A

Y

S

27

CAGTAGAGAAACACAGGAGCTGATTGATCAACAATTGTATTAAAAAGTAATCATGTAGCC TTCATCGCATGCATCCATAGTGTTTCTCTATCTATTGACAGGGAAGCAGATGCATTTGTT GCACTGAGGCCATGATCCTGTTCTCTGTTTGACTTCTCTCTAAAGGTGAGAGGGCATCAC G

E

R

A

S

T

M

A

K

T

D

A

G

R

M

E

K

Y

R

S

K

I

N

S

G

A

K

Y

G

I

D

P

A

L

I

A

A

I

I

S

R

E

S

A

G

N

A

L

H

D

G

W

G

D

Y

D

S

K

R

G

A

Y

G

W

G

L

M

Q

V

D

V

N

G

G

G

H

T

A

Q

G

A

W

D

S

E

E

H

L

R

CAAGGCACCGAGATCTTGGTTCATTTTATCAATCGGATCCGCAACAAATTTCCTGGCTGG Q

G

T

E

I

L

V

H

F

I

N

R

I

R

N

K

F

P

G

840 102

AATCCAAACGGAGGTGGACACACTGCACAGGGCGCATGGGACAGTGAGGAACACCTCCGC P

780 99

AGAGCCTCACATTACAATACAATAACAGCAGTGTTCATCCCATTTCTGTAGGTTGATGTT

N

720 92

ACGGCTGGGGACTGATGCAGGTTGGAAGAAATCTGCTGTTTAACTCACATCCTCTTAGAT N

660 72

GGGCCGGAAATGCCCTACATGATGGCTGGGGAGACTATGACTCAAAGAGAGGAGCGTATA R

600 52

TGGGAGCTAAATATGGAATCGATCCAGCTCTAATCGCTGCCATCATCTCCAGAGAGTCCA V

420 480 540 32

ACACCATGGCAAAGACTGATGCGGGCAGAATGGAAAAGTACAGGTCTAAAATCAACAGTG H

12

360

W

900 122

960 142

AGCACGGAGCAGCAGCTGAAAGGTTTGTTTCACTGAACCAGTAGACTCGCACATGCACCA 1020 S

T

E

Q

Q

L

K

149

ACTCACTTCACACTGTTTAGCTTCGTTTGAATTTGTTTTTGTCTCTGCAGGAGGGATAGC 1080 G

G

I

A

153

AGCTTACAATATGGGGGATGGAAACGTCCATTCCTATGAAAACGTGGATGAGAACACAAC 1140 A

Y

N

M

G

D

G

N

V

H

S

Y

E

N

V

D

E

N

T

T

173

AGGTAAAGACTACTCCAATGACGTCGTTGCCAGAGCTCAGTGGTACAAAAACAACGAAGG 1200 G

K

D

Y

S

N

D

V

V

A

R

A

Q

W

Y

K

N

N

E

G

193

CTTTTAACAGCTGAAGCTGTCCACGACAAAATTCTCTGAAAAATCTGCAAATCATGAAGG 1260 F

Stop

AAACACTATGTAATGTGTGCTAAATAAACATAATGCAAAGCAAATCC

194

1307

Fig. 1. Compiled genomic organization of the mandarin fish g-lysozyme gene (Genbank accession number: AY738131). Exons are in bold and introns are in italic; the translation of the exon-coding regions is also given. The start and stop codons for translation and the splicing sites between exons and introns are shaded. The polyadenylation signal is underlined.

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2.4. Lytic activity assays The lytic activity assay was performed using Micrococcus lysodeikticus as substrate according to the method of Hikima et al. (2001). M. lysodeikticus in 1% warm (50 °C) melting agarose (50 mM phosphate buffer, pH 6.2) was poured onto plates. The concentration of M. lysodeikticus was adjusted to 0.2 OD at 600 nm, which is equivalent to 100 μg/ml. The recombinant mandarin fish lysozyme (250 μg), hen egg-white lysozyme (250 μg, Sigma) and negative control were put in individual wells in the agarose plates and incubated at 30 °C for 24 h. 3. Results 3.1. Characterization of the mandarin fish g-lysozyme gene The mandarin fish g-lysozyme mRNA has 742 nucleotides (nt) in length, and contains an open reading frame of 582 nt encoding 194 amino acids, with 57 nt located in the 5′ untranslated region (UTR) and 103 in

109

the 3′ UTR (Fig. 1). The polyadenylation signal was found 19 nt before the poly A signal. In the 3′ UTR, no unstable motifs of ATTTA were found. From the first transcription initiation site, the mandarin fish g-lysozyme gene extends 1307 nt to the end of the 3′ UTR, and it contains 5 exons and 4 introns. All the 5′ and 3′ ends of the introns show canonical splicing motifs (GT/intron/AG). The gene structure of mandarin fish g-lysozyme was quite similar to its homologue in Japanese flounder. The two genes consist of 5 exons and 4 introns, with exons 1∼4 each encoding the same number of amino acids and having the same splicing sites. The only difference exists in the 5th exon, with only one aa longer at the end in flounder. When compared with homologues from other species of fish (Fig. 2), the aa sequence of the mandarin fish glysozyme shared 86% identity with orange-spotted grouper, 75% with Japanese flounder, 59% with puffer fish and 57% with zebrafish. The three catalytic residues (Glu73, Asp86 and Asp97 in mandarin fish) and their neighboring amino

Ec

1 -------------MGYGNIMNVETTGASWQTAQQDKLGYSGVRASHTMANTDSGRMERYR

Sc

1 -------------MGYGNIMRLETTGASWETAQQDSLAYSGERASHTMAKTDAGRMEKYR

Po

1 -------------MSYGQIRLVETSGASGATSQQDNLGYSGVKASHKMAEIDSGRMSKYK

Tr

1 -------------MPYGKIEDIKTSGASDVTAAQDGLKEGGWKSSHRMAEIDSNRMENYR

Dr

1 MGIPVILTMYFLACIYGDIMKIDTTGASEVTAKQDKLTVKGVEASKKLAEHDLARMEQYK

Ec

48 SKINSVGAKYGIDPALIAAIISEESRAGNVLHDGWGDYDSNRGAYNAWGLMQVDVNP--N

Sc

48 SKINSVGAKYGIDPALIAAIISRESRAGNALHDGWGDYDSKRGAYNGWGLMQVDVNP--N

Po

48 SKINKVGQSYGIEPALIAAIISRESRAGNQLKDGWGDWNPQRQAYNAWGLMQVDVNP--N

Tr

48 TIINEAGRQCDVDPAVIAGIISRESRAGNQLINGWGDHG------KAFGLMQIDVTPPPN

Dr

61 SKILKVARAKQMDPAVIAAIISRESRAGAALKDGWGDHG------NGFGLMQVDKRY---

Ec

106 GGGHTARGAWDSEEHLSQGAEILVYFIGRIRNKFPGWNTEQQLKGGIAAYNMGDGNVHSY

Sc

106 GGGHTAQGAWDSEEHLRQGTEILVHFINRIRNKFPGWSTEQQLKGGIAAYNMGDGNVHSY

Po

106 GGGHTAVGGWDSEDHLRQATGILVTFIERIRTKFPGWSKEKQLKGGIAAYNMGDKNVHSY

Tr

102 GGGHTPVGTWDSLEHLIQATEILVEFIERIKTKFPRWNADQHLK-ALAAYNKGEKNVESY

Dr

112 ---HKLVGAWDSEEHLTQGTEILIGYIKDIKAKFPTWTKEQCFKGGISAYNAGVKNVQTY

Ec

166 DNVDGRTTGGDYSNDVVARAQWYKTQKGF-

Sc

166 ENVDENTTGKDYSNDVVARAQWYKNNEGF-

Po

166 EGVDENTTGRDYSNDVTARAQWYRDNGYSG

. **.*. ..*.*** *..** *...*...*...*. *..**..*.

..*. ..

....**.**.***.*****. *..****.

. .....****.*...

.

...*...*.***..** *...**. .* .*. ***.*. ....*....***.*..**..*

Tr

161 ASVDAKTTGKDYSNDVVARAQWYKSNMGF-

Dr

169 ERMDVGTTGGDYANDVVARAQWFKSKGY-. .*

***.**.***.*****.... ..

Fig. 2. Amino acid sequence alignment of mandarin fish g-lysozyme with the sequences from other species of fishes obtained using Clustal W program. The Genbank accession numbers were: Japanese flounder (Po) AB050590; pufferfish (Tr) AB126244; zebrafish (Dr) NM001002706; orange-spotted grouper (Ec) AF416458. Asterisks (*) represent identity, and dots (·) conservative substitution.

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acids were conserved in fish. No signal peptide was predicted to exist at the N-terminus of all fish g-lysozyme. 3.2. Comparison of the 5′ flanking region of g-lysozyme genes between mandarin fish and Japanese flounder About 560 bp in the flanking region of g-lysozyme genes were compared between mandarin fish and Japanese flounder (Fig. 3), and several comparatively conserved regions and putative transcriptional factor binding sites could be found. A putative ISCBP (IRF8) binding site was located at − 40∼−49 in mandarin

fish and −44∼− 53 in the flounder. One cluster of binding sites for Oct-1, AP4 and C/EBPα was detected − 127∼− 144 in mandarin fish and − 127∼− 144 in flounder, and another for Oct-1 and C/EBPα found − 242∼− 254 in mandarin fish and − 273∼− 286 in flounder. A relatively conserved NFκB binding site found in − 407∼− 416 in mandarin fish was located at − 402∼− 411 in flounder. Several other differences were also found in the flanking region. TATA box found at 30 bp upstream in flounder does not exist in mandarin fish, while more putative NFκB and ISCBP (IRF-8) binding sites were present in the latter.

Fig. 3. The comparison of 5′ flanking region of the g-lysozyme gene between mandarin fish (Sc) and Japanese flounder (Po). The putative NFκB binding sites are shaded, and putative ICSBP (IRF8) binding sites are boxed. C/EBPα, Ap1, Ap4, Hb binding sites and TATA box are underlined. Asterisks (*) represent identity. The position +1 of the nucleotide sequence is defined as the first nucleotide of exon 1. The TRANSFAC database was used to search for putative binding sites of transcription factors.

B.J. Sun et al. / Aquaculture 252 (2006) 106–113

111

20.1

NTA column with buffer containing 250 mM imidazole (Fig. 4). As a fusion protein, the recombinant g-lysozyme contained a stretch of 122 aa residues at the N-terminus, allowing enhanced solubility and affinity purification. The size of the purified g-lysozyme (approximately 35 kDa) matched well with the combined mass of the natural g-lysozyme (21.3 kDa) and the inserted fragment (13.4 kDa). The purified recombinant g-lysozyme caused lysis of Micrococcus lysodeikticus at pH 6.2, 30 °C (Fig. 5), but the recombinant g-lysozyme could only lyse a narrow circle while the same dose of hen egg c-lysozyme could lyse a circle with 3 cm in diameter.

14.4

4. Discussion

kDa 97.4 66.2 45.0

31.0

1

2

3

4

5

Fig. 4. Expression and purification of mandarin fish recombinant glysozyme in E. coli cells. The collected cell lysates and the purified proteins were separated on 12% SDS-PAGE reducing gels. Lane 1: protein molecular standard; 2: pET-32a without insert; 3: pET-32alysozyme without IPTG induction; 4: pET-32a-lysozyme with IPTG induction; 5: purified recombinant lysozyme.

3.3. Expression of the recombinant g-lysozyme in bacteria and assay of its lytic activity A high level expression was observed in DE3 cells transformed with pET-32a-lysozyme with IPTG induction when cultured between 20 and 37 °C, and after gentle sonication on ice, the recombinant lysozyme was released into the supernatant. The recombinant protein could be eluted specifically from the Ni–

A

B

The same gene structure and splicing sites of glysozyme in mandarin fish and Japanese flounder may indicate that g-lysozyme is a conserved molecule in teleosts, since their taxonomical status were quite different, with the mandarin fish belonging to Perciformes, while the Japanese flounder to Pleuronectiformes. The g-lysozyme gene pattern in fish is quite different from that in avian species, as the latter has a gene structure of 6 exons and 5 introns (Nakano and Graf, 1991). The N-terminal signal peptide found in birds and mammals which leads to secretion is absent in fish, and it has been suggested by Irwin and Gong (2003) that fish g-lysozyme may not be a secreted protein because of the absence of the signal peptide and its wide expression in various tissues. The high similarity of the amino acid sequences of fish g-lysozymes may indicate that fish g-lysozymes

C

Fig. 5. Lytic activity of recombinant mandarin fish g-type and hen egg-white c-type lysozyme against Micrococcus lysodeikticus. Small central circles are the wells containing the samples; larger circles represent lysed halos formed by the lysozyme on M. lysodeikticus substrate. A: control; B: purified recombinant g-lysozyme; C: hen egg-white lysozyme.

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have an important role in defense, as three residues, i.e. Glu73, Asp86 and Asp97, which are thought to have strong catalytic activity for bird egg g-lysozyme (Weaver et al., 1985) have been conserved in all available fish g-lysozyme sequences. It is interesting to note that some common transcriptional factor binding sites exist in the flanking region of the mandarin fish and also Japanese flounder g-lysozyme genes, such as ISCBP, NFκB, Oct-1, AP4 and C/EBPα. Since a functionally similar protein in different species may have similar regulatory mechanisms, the putative transcriptional factor binding sites found in the mandarin fish may actually be the real ones. Several C/EBPα binding sites found in the mandarin fish g-lysozyme sequence are universal elements, and the NFκB and C/EBP co-operative binding pattern has been found in promoters of many other immune genes (Shelest et al., 2003). Heterocomplexes containing ICSBP may function predominantly in activating transcriptional complexes (Mamane et al., 1999), and it has been shown that cooperative interactions between ICSBP, PU.1 and IRF-1 may increase the expression of the gp91phox gene encoding a subunit of the phagocyte respiratory burst oxidase catalytic unit (Eklund et al., 1998). The Oct-1 binding sites are ubiquitous in some immune gene promoters, seen as in the granulocyte/macrophage colony stimulating factor, IL-3, and IL-5 promoters where the Oct1 contributes to gene transcription, but Oct-1 and C/EBP bind to overlapping elements within the interleukin8 promoter to repress gene transcription (Wu et al., 1997; Kaushansky et al., 1994). The existence of ISCBP and NFκB binding sites may indicate that the transcription of fish g-lysozyme is influenced by other immune genes, since they are immune-related transcriptional factors, but further experiments are needed to clarify the exact mechanism involved. The pET system is one of the most powerful ones yet developed for the cloning and expression of recombinant proteins in E. coli. Sometimes the recombinant protein could reach as high as 90% of the total protein (LaVallie et al., 1993), and the recombinant mandarin fish glysozyme accumulated in the E. coli cytoplasm at a relatively high level. Although a stretch of 122 aa residues were added to the N-terminus of the recombinant protein, it still has the biological activity, just like several mammalian cytokines and growth factors expressed in this system (LaVallie et al., 1993). Since a high percentage of recombinant protein was present in the cytoplasm, it could be simply released from E. coli cytoplasm by freezing/thawing treatments and purified by convenient steps. The recombinant

flounder c-type and g-type lysozymes have been constructed using the baculovirus expression system with Sf21 (Spodoptera frugiperda 21) insect cells, with their lytic activity proved (Hikima et al., 2001). The recombinant flounder c-type lysozyme showed a relatively higher level of lytic ability than the g-type lysozyme, which was even higher than commercial hen egg white lysozyme (Hikima et al., 2001). The recombinant mandarin fish g-lysozyme expressed in E. coli should be analyzed in further research in relation to its ability to lyse bacteria, especially pathogenic ones, and its application in industrial production should be evaluated. Acknowledgements This study was supported by grants (project nos. 30130150 and 30025035) from the National Natural Science Foundation of China, partially by a project (KSCX2-SW-302) from Bureau of Life Science and Biotechnology, Chinese Academy of Sciences (CAS), and by a project (220312) from Institute of Hydrobiology, CAS. References Canfield, R.E., McMurry, S., 1967. Purification and characterization of a lysozyme from goose egg white. Biochem. Biophys. Res. Commun. 26, 38–42. Dautigny, A., Prager, E.M., Pham-Dinh, D., Jolles, J., Pakdel, F., Grinde, B., Jollès, P., 1991. cDNA and amino acid sequences of rainbow trout (Oncorhynchus mykiss) lysozymes and their implications for the evolution of lysozyme and lactalbumin. J. Mol. Evol. 32, 187–198. Eklund, E.A., Jalava, A., Kakar, R., 1998. PU.1, IRF-1, and ICSBP cooperate to increase gp91 phox expression. J. Biol. Chem. 273, 13957–13965. He, J.G., Zeng, K., Weng, S.P., Chan, S.M., 2002. Experimental transmission, pathogenicity and physical-chemical properties of infectious spleen and kidney necrosis virus (ISKNV). Aquaculture 204, 11–24. Hikima, J., Hirono, I., Aoki, T., 2000. Molecular cloning and novel repeated sequences of a C-type lysozyme gene in Japanese flounder (Paralichthys olivaceus). Mar. Biotechnol. 2, 241–247. Hikima, J., Minagawa, S., Hirono, I., Aoki, T., 2001. Molecular cloning, expression and evolution of the Japanese flounder goosetype lysozyme gene, and the lytic activity of its recombinant protein. Biochim. Biophys. Acta 1520, 35–44. Irwin, D.M., Gong, Z.Y., 2003. Molecular evolution of vertebrate goose-type lysozyme genes. J. Mol. Evol. 56, 234–242. Jollés, P., Jollés, J., 1984. What is new in lysozyme research? Mol. Cell. Biochem. 63, 165–189. Kaushansky, K., Shoemaker, S.G., O'Rork, C.A., McCarty, J.M., 1994. Coordinate regulation of multiple human lymphokine genes by Oct-1 and potentially novel 45 and 43 kDa polypeptides. J. Immunol. 152, 1812–1820.

B.J. Sun et al. / Aquaculture 252 (2006) 106–113 LaVallie, E.R., DiBlasio, E.A., Kovacic, S., Grant, K.L., Schendel, P.F., McCoy, J.M., 1993. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology 11, 187–193. Liu, J., Cui, Y., Liu, J., 1998. Food consumption and growth of two piscivorous fishes, the mandarin fish and the Chinese snakehead. J. Fish Biol. 53, 1071–1083. Mamane, Y., Heylbroeck, C., Génin, P., Algarté, M., Servant, M.J., LePage, C., DeLuca, C., Kwon, H., Lin, R.T., Hiscott, J., 1999. Interferon regulatory factors: the next generation. Gene 237, 1–14. Nakano, T., Graf, T., 1991. Goose-type lysozyme gene of the chicken: sequence, genomic organization and expression reveals major differences to chicken-type lysozyme gene. Biochim. Biophys. Acta 1090, 273–276. Nielsen, H., Engelbrecht, J., Brunak, S., Von Heijne, G., 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6. Prager, E.M., Jollès, P., 1996. Animal lysozymes c and g: an overview. In: Jollès, P. (Ed.), Lysozymes: Model Enzymes in Biochemistry and Biology. Birkhäuser Verlag, Basel, pp. 9–31. Shelest, E., Kel, A., Göβling, E., Wingender, E., 2003. Prediction of potential C/EBP/NFκB composite elements using matrix-based search methods. In Silico Biol. 3, 0007.

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Sun, B.J., Nie, P., 2004. Molecular cloning of the viperin gene and its promoter region from the mandarin fish Siniperca chuatsi. Vet. Immunol. Immunopathol. 101, 161–170. Weaver, L.H., Grütter, M.G., Remington, S.J., Gray, T.M., Isaacs, N.W., Matthews, B.W., 1985. Comparison of goose-type, chicken-type, and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution. J. Mol. Evol. 21, 97–111. Wingender, E., Chen, X., Hehl, R., Karas, H., Liebich, I., Matys, V., Meinhardt, T., Prüβ, M., Reuter, I., Schacherer, F., 2000. TRANSFAC: an integrated system for gene expression regulation. Nucleic Acids Res. 28, 316–319. Wu, G.D., Lai, E.J., Huang, N., Wen, X., 1997. Oct-1 and CCAAT/ enhancer-binding protein (C/EBP) bind to overlapping elements within the interleukin-8 promoter: the role of Oct-1 as a transcriptional repressor. J. Biol. Chem. 272, 2396–2403. Yin, Z.X., He, J.G., Deng, W.X., Chan, S.M., 2003. Molecular cloning, expression of orange-spotted grouper goose-type lysozyme cDNA, and lytic activity of its recombinant protein. Dis. Aquat. Org. 55, 117–123. Zhang, Y.A., Nie, P., Wang, Y.P., Zhu, Z.Y., 2003. cDNA sequence encoding immunoglobulin M heavy chain of the mandarin fish Siniperca chuatsi. Fish Shellfish Immunol. 14, 477–480.