ANNUAL REPORT - Department of Biotechnology

model. The seventh fraction of the PVF contained
cardiac development promoting activity. Sequencing
and identification of active protein molecules was
done.
Due to possible relationship between cardiac
development and angiogenesis, preliminary
experiments to assess the angiogenic potential of
PVF were carried out. Transcriptional studies were
carried out by quantitative real-time PCR for the
markers Noggin (Signalling molecule, antagonistic to
BMP2, recently shown to be involved in
cardiomyogenic differentiation). There was overall
increase in the expression of these markers in chick
th th
embryo of the 5 stage of development. At 7 Stage of
development there was a decrease in their overall
expression and this trend was reversed in stage 10th
of development of chick embryo. Perivitelline fluid of
the eggs of the horse shoe crab was fractionated and
th
7 fraction was found to possess early cardiac
development promoting activity.
Cell Lines from Seabass
A project was continued to develop cell lines from sea
bass (Lates calcarifer). Kidney and spleen cell lines
from sea bass were established and designated as
SISK and SISS. These lines have been deposited in
NCCS, Pune. The established cell lines have been
used for isolation of nodavirus which is responsible
for viral nervous necrosis (VNN) in sea bass larvae.
The nodavirus was isolated from infected sea bass
(Lates calcarifer) larvae during a massive outbreak in
sea bass hatcheries using these cell lines. The typical
cytopathic effect of nodavirus was characterized by
multiple vacuolations was observed in SISK.
Embryonic stem cell cultures were initiated from
blastula stage embryo of sea bass. The sea bass
embryonic cells were small, round or polygonal with a
big nuclei and sparse cytoplasm in shape.
Differentiation of embryonic cells into variety of cell
types was induced by all-tansretinoic acid and
differentiated into various cell types, including
neuron-like cells, oligodentritic, muscle cells and
93
unidentified cells.
Extensive CPE with multiple vacuolation (arrow) in SISK cells infected
with nodavirus
Shrimp Genomics
Expression of immune function associated proteins
from shrimp Penaeus monodon was studied at
College of Fisheries Mangalore, Central Institute of
Brackishwater Aquaculture Chennai, Anna
University Chennai and National Institute of
Oceanography Goa. The gene coding for a c-type
lysozyme from P. monodon was amplified by reverse
transcriptase PCR after the animals were challenged
with luminous bacterial pathogen, Vibrio harveyi.
The amplified DNA fragment was cloned in pEXP5-
NT/TOPO expression vector and recombinant
plasmids were transformed into competent BL21
Escherichia coli cells. Lysozyme was over
expressed by induction with 1 mM IPTG. The
recombinant protein was found to be active against
both gram positive bacteria (Micrococcus luteus)
and garm negative bacteria (Vibrio harveyi). The
commonly used lysozyme standard from hens egg
white is active only against gram negative bacteria.
A similar collaborative project was undertaken at
Cochin University of Science and Technology on
development and application of CMG family
recombinant hormones, their antagonists and RNAi
technique for induced maturation and spawning of
Penaeus monodon. Sequence data available with
the genbank was utilized to design primers to get
ORF amplified. The amplified product showed a 98%
Research and Development