Targeted disease prophylaxis in European fish farming (TargetFish)

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frame (ORF) that encodes 196 amino acids. The 2-cys Prx signature motif 1
(FYPLDFTFVCPTEI) and motif 2 (GEVCPA) were conserved in CpPrx amino
acid sequences, Sequence comparison showed that CpPrx shares 67–74 %
identities with known species Prx1 or Prx2, phylogenetic tree indicated
that CpPrx belongs to typical 2-Cys Prx. This indicated that it was a
member of 2-Cys Prx. Expression of CpPrx was detected in organs of C.
plicata, such as haemocyte, mantle, adductor muscle, hepatopancreas and
gill, with the highest expression levels found in gill and hepatopancreas.
The expression levels of CpPrx significantly increased in hepatopancreas at
6, 12 and 24 h after injection by Aeromonas hydrophila (6h, P<0.05; 12h,
P<0.01; 24h, P<0.05), and resumed to normal levels at 48 h. In addition,
the CpPrx gene was subcloned into a vector pET-32a (+) and transformed
into Escherichia coli BL21 (DE3), the recombinant plasmid was expressed
successfully by SDS-PAGE analysis. The CpTPx coding sequences in pET-
32a(+) were transformed into the cells of E. coli, the result showed that it
could resist to H
2
O
2
at 0w0.8 mM concentration.
*Corresponding author.
E-mail address: cgwen@ncu.edu.cn (C.G. Wen)
P-296.
Targeted disease prophylaxis in European fish farming (TargetFish)
G. Wiegertjes
1
,
*
, G. Lorenzen
2
, C. Secombes
3
, B. Collet
4
, U. Fischer
5
,
C. Tafalla
6
, D. Parra
7
, G. Scapigliati
8
, P. Boudinot
9
, Ø. Evensen
10
,
A. Adams
11
, A. Toffan
12
, K. Buchmann
13
, T. Vesely
14
, L. David
15
,
V. Mulero
16
, P. Smith
17
, V. Aspehaug
18
, K. Engell-Sørensen
19
, J. Sober
20
,
T. Wallis
21
, T. Rød
22
, M. Flores
23
, J. March
24
, A. Stratmann
25
,
P. Christofilogiannis
26
, J. Tobar
27
, N.H. Henriksen
28
, T. Sigholt
29
,
A. de las Heras
30
.
1
Wageningen University, The Netherlands;
2
Technical University, Denmark;
3
University of Aberdeen, United Kingdom;
4
Marine Scotland, United Kingdom;
5
Friedrich Löffler Institut, Germany;
6
Instituto Nacional De Investigacion Y Tecnologia Agraria Y
Alimentaria, Spain;
7
Universitat Autonoma de Barcelona, Spain;
8
Università degli Studi della Tuscia, Viterbo, Italy;
9
Institut National De La Recherche Agronomique, France;
10
Norwegian School of Veterinary Science, Norway;
11
The University of Stirling, United Kingdom;
12
Istituto Zooprofilattico Sperimentale delle Venezie, Italy;
13
Københavns Universitet, Denmark;
14
Veterinary Research Institute, Czech Republic;
15
The Hebrew University of Jerusalem, Israel;
16
University of Murcia, Spain;
17
Tethys Aquaculture Limited, Greece;
18
PatoGen Analyses AS, Norway;
19
Fishlab, Denmark;
20
Naxo OÜ, Estonia;
21
Ridgeway Biologicals Limited, United Kingdom;
22
Rossi A/S, Denmark;
23
Ingeniatrics Tecnologias S.L., Spain;
24
BigDNA, United Kingdom;
25
W42 Industrial Biotechnology GmbH, Germany;
26
AQUARK, Greece;
27
CentroVet, Chile;
28
Danish Trout Association, Denmark;
29
BioMar A/S, Denmark;
30
Bionaturis, Spain
Abstract
TargetFish is a large collaborative project funded by the European Com-
mission under the 7th Framework Programme for Research and Techno-
logical Development (FP7) of the European Union (Grant Agreement
311993 TARGETFISH). The project started November 2012 and will run for
5 years, bringing together leading European research groups that are ex-
perts on the fish immune system and enterprises from the Biotech and
Veterinary sectors that aim to commercialize fish vaccines for European
fish farming. TargetFish will advance the development of existing (but
insufficient) and new prototype vaccines against socio-economically
important viral or bacterial pathogens of Atlantic salmon, rainbow trout,
common carp, sea bass, seabream and turbot. TargetFish will also establish
a knowledge- and technology-base for rational development of next
generation fish vaccines. The project will develop targeted vaccination
strategies for currently sub-optimal and for novel vaccines. Improved
vaccines will be brought closer to industrial application by addressing
practical issues such as efficacy, safety and delivery route. The main ob-
jectives of the project are to: 1) generate knowledge by studying antigens
and adjuvants for mucosal routes of administration while analyzing the
underpinning protective immune mechanisms; 2) validate this knowledge
with response assays for monitoring vaccine efficacy and safety, including
issues associated with DNA vaccines; 3) approach implementation of
prototype vaccines by optimizing vaccination strategies thus 4) shortening
the route to exploitation. To achieve these challenging tasks, we brought
together 30 partners from 10 EU member states, 2 associated countries and
1 International Cooperation Partner Country (ICPC). In this large multi-
disciplinary consortium an approximate equal number of RTD and SME
partners will cooperate closely while keeping an intensive communication
with the large vaccine and nutrition industries via an Industry Forum.
*Corresponding author.
E-mail address: geert.wiegertjes@wur.nl (G. Wiegertjes)
P-170.
Expression analysis of i-type lysozyme gene from Cristaria plicata and
enzymatic activity analysis
D. Wu, C.G. Wen
*
, B.Q. Hu, X.J. Hu, Z.Y. Tao.
School of Life Sciences and Food Engineering, Institute of Life Science,
Nanchang University, Nanchang 330031, China
Abstract
Two i-type lysozymes (CpLYZ1, CpLYZ2) cDNAs were cloned from fresh-
water mussel, Cristaria plicata, using rapid amplification of cDNA ends and
nested PCR.
The full-length cDNA sequence of CpLYZ1 was 763bp (GenBank accession
number: JQ988052), consistingof a50-terminal untranslated region (UTR) of
21 bp, a 30-terminal UTRof 259 bp with a 29 bp poly (A) tail, a tailing signal
(AATAAA) anda ATTTA (RNA instability motifs), and the open reading frame
of 483bp. The CpLYZ1 cDNA encoded a polypeptide of 160 amino acids with
a predicted molecular mass of 17.8 kDa, anda theoretical isoelectric point of
6.07. The N-terminus had the features consistent with a signal peptide as
defined by SignalP software with a putative cleavage site located after po-
sition 23 (MEAVKMFFFLVGLLLTFNKCAES), the deduced mature peptide was
of 137 amino acid residues, and the mature peptide harbored fourteen
cysteine residues. The deduced amino sequence of CpLYZ1 contains two
highly conserved i-type lysozyme activity sites (Glu61 and Ser72); i-type
specific motif: C-L-[E/L/R/H]-C-[I/M]-C; and the conserved motif
MDVGSLSCG(P/Y)(Y/F)QIK in bivalve i-type lysozymes.The full-length cDNA
sequence of CpLYZ2 was 913bp (GenBank accession number: JQ988051),
consisting of a 50-terminal untranslatedregion (UTR) of 38 bp, a 30- terminal
UTR of 389 bp with a 29 bp poly (A) tail, a tailing signal (AATAAA) and a
ATTTA (RNA instability motifs), and the open reading frame of 486bp.The
CpLYZ2 cDNA encoded a polypeptide of 161 amino acids with a predicted
molecular mass of 18.2kDa, and a theoretical isoelectric point of 6.56. The N-
terminus had the features consistent with a signal peptide as defined by
SignalP software with a putative cleavage site located after position 23
(MEAVKMFFFLVGVLLTFTKCTES), the deduced mature peptide was of 138
amino acid residues, and the mature peptide harbored twelve cysteine
residues. The deduced amino sequence of CpLYZ2 contains only one highly
conserved i-type lysozyme activity sites (Glu61).It has i-type specific motif:
C-L-[E/L/R/H]-C-[I/M]-C, and the part of the conserved sequence of amino
acids in bivalve i-type lysozymes.The expression of CpLYZ1 mRNA and
CpLYZ2 mRNA in Cristaria plicata were measured by fluorescent real-time
quantitative RT-PCR. The results showed that the mRNA expression of
CpLYZ1 and CpLYZ2 had significant differences in the organization. The
Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–17521746