Faculty of Science - Mahidol University

340
STRUCTURAL AND ANTIGENIC ANALYSIS OF
THE YELLOW HEAD VIRUS NUCLEOCAPSID
PROTEIN p20 (NO. 904)
Nusra Sittidilokratna 1,2 , Natthida Phetchampai 1,2 , Vichai
Boonsaeng 2 and Peter J Walker 3
1 National Center for Genetic Engineering and Biotechnology
(BIOTEC), National Science and Technology Development
Agency (NSTDA), Phathumthani 12120, Thailand; 2 Center
Excellence for Shrimp Molecular Biology and Biotechnology,
Faculty of Science, Mahidol University, Bangkok 10400,
Thailand; 3 CSIRO Livestock Industries, Australian Animal
Health Laboratory, Bictora 3220, Australia.
Key words : Yellow head virus, shrimp virus, nucleoprotein,
bacterial expression
Yellow head virus (YHV) is an invertebrate nidovirus that
is highly pathogenic for marine shrimp. Nucleotide sequence
analysis indicated that the YHV ORF2 gene encodes a basic protein
(pl = 9.9) of 146 amiono acids with a predicted molecular weight of
16,325.5 Da. The deduced amino acid sequence indicated a
predominance of basic (15.1%) acidic (9.6%) and hydrophilic polar
(34.3%) residues and high proportion proline and glycine residues
(16.4%). The ORF2 gene was cloned and expressed in E. coli as a
M r = 21 kDa His 6 - protein that reacted with YHV nucleoprotein
(p20) monoclonal antibody. Segments representing the four linear
quandrants of the nucleoprotein were also expressed in E. coli as
GST –fusion proteins. Immunoblot analysis using YHV polyclonal
rabbit antiserum indicated the presence of linear epitopes in all except
the V 37 –Q 74 quadrant. Immunoblot analysis of the GST-fusion
proteins and C-terminally truncated segments of the nucleoprotein
allowed mapping of YHV monoclonal antibodies Y19, Y20 and YII4
to linear epitopes in the acidic domain between amino acids I 116 and
E 137 . The full-length nucleoprotein was expressed at high level in
E.coli and was easily purified in quantity from the soluble cell
fraction by Ni + - NTA affinity chromatography.
BACTERIAL CLEARANCE RATE AND A NEW
DIFFERENTIAL HEMOCYTE STAINING METHOD
TO ASSESS IMMUNOSTIMULANT ACTIVITY ION
SHRIMP (NO. 905)
Kallaya Sritunyalucksana 1 , Warachin Gangnongiw 1 , Somwit
Archakunakorn 2 , Daniel Fegen ,Timothy W. Flegel 1
1 National Center for Gentic Engineering and Biotechnology
(BIOTEC) and Centex Shrimp , and 2 Shrimp Biotecnology
Business Unit (SBBU), Faculty of Science, Mahidal University,
Rama VIRoad, Bangkok 10400, Thailand.
Key words : Black tigr shrimp Penaeus⋅ mondon⋅
Hemocytes⋅Bacterial clearance⋅Immumostimulant
New methods were developed to assess immunostimulant
efficacy in the black tiger shrimp Penaeus monodon. Test Shrimp
were fed with 2 or 4 % yeast extract (YE)-coated feed while controls
were fed non-coated feed. After 4 wk of feeding , individual shrimp
were assessed for total hemocvte counts(THC), the number of
granular hemocytes (GH) and rate of bacterial clearance . For
hemocyte counts, formalin-fixed hemolymph was stained with 1.2
% Rose Bengal in 50 % ethanol for 20 min at room temperature .
Some of this mixture was used for THC with a hemocytometer
while some was smeared on a microscope slide and left to day before
counterstaining with hematoxylin for GH counts , By this tecnicque
,high quality smears were obtained for accurate differential
counts.Bacterial clearance assays were used to assess the sum effect
of humoral and cellular defense mechanisms. Vibrio harveyi was
injected intramuscularly at 1x 10 8 cells per shrimp and hemlymph
was collected in anticoaqulant 0,15,30 and 60 min post-injection
for quadruplicate drop count (20 μl) on TCBS agar.Total hmocyte
counts for shrimp fed with 4% YE were singnificantly higher
(p