Bariloche protein symposium argentine society for biochemistry and molecular biology

74
BIOCELL, 27 (Suppl. I), 2003
MI-P58.
CHARACTERIZATION OF ROTAVIRUS VP6 PROTEIN AS
CARRIER TO HETEROLOGOUS EPITOPES
Peralta Andrea, Lopez M. Gabriela, Taboga Oscar and Carrillo
Elisa.
Instituto de Biotecnología, CICVyA, INTA Castelar. E-mail:
[e-mail]
The general goal of this project is to evaluate the use of virus like
particles (VLPs) based on VP6 protein from simian rotavirus as
presentation system for heterologous antigens. Due to the spatial
nature of VLPs, the antigenic sites are presented to the immune
system in a multimeric way, associated to immunogenic proteins.
Indeed, VLPs do not replicate and are non-infectious vehicles. In
order to investigate the putative sites for insertion of foreign
sequences into VP6 protein, the 14 amino-acid epitope V5 was
chosen. Six putative sites for insertions were selected comprising
amino-acidic positions 14, 101, 146, 235, 382 and the amino
terminus. Recombinant baculovirus were constructed and
chimaeras VP6-V5 were expressed in insect cells, rendering high
expression levels all of the assessed versions. Version VP6
14
 was
able to form trimers, but it was not recognized by a polyclonal
rabbit serum anti-rotavirus, whereas an anti-VP6 monoclonal
antibody strongly reacted with VP6
14
 , indicating that a major
antigenic site into VP6 is being interrupted by the insertion. Version
VP6
235
  was not able to form trimers, whereas the rest of the
versions exhibited trimer formation and were recognized by both
the rabbit serum anti-rotavirus and the monoclonal antibody anti-
V5.  Immunogenicity of the different chimaeras is being evaluated.
MI-P59.
EVALUATION OF ANTIGENICITY OF THE
RECOMBINANT E2 GLYCOPROTEIN OF CLASSICAL
SWINE FEVER VIRUS
Pereda A, Miquet J, Taboga O, and Piccone M.
Instituto de Biotecnología, INTA Castelar. E-mail:
[e-mail]
Classical swine fever (CSF) is a highly contagious disease that
affects pigs, causing great economic losses. Argentina uses a
lapinized chinese isolate for vaccination and the vaccinated animals
cannot be differentiated by serology from those infected. Obviously,
a vaccine that allows the discrimination between vaccinated and
infected pigs is essential to eradicate this disease.
CSF virus genome consists on a molecule of RNA of positive
polarity with a single open reading frame coding for a polyprotein
flanked by highly conserved non-coding regions. This polyprotein
is processed by viral and cellular proteases to generate structural
and nonstructural proteins. The infection with the CSFV stimulates
the production of antibodies against the proteins E2, Erns and
NS3. Nevertheless, only E2 induces the production of neutralizing
antibodies in pigs. This fact reveals that a vaccine based on E2
would allow to discriminate those animals vaccinated from those
infected. The purpose of this work was the expression of the protein
E2 in procariotic and eucariotic systems to be used either as a
vaccine or as a diagnostic reagent. The genomic region that codes
for the E2 glycoprotein was amplified by RT-PCR and cloned in
vectors for expression in E. coli BL21 and in the system
baculovirus/ insect cells. Both vectors incorporate a 6 histidine
residues (His
6
) in the NH
3
 terminal end of the E2 to allow the
purification. In tests of western-blot, purified proteins were
revealed with Mab against CSFV E2, Mab anti-His
6
 and with
serum from infected and non infected pigs.
MI-P60.
PHOTOPROTECTIVE COMPOUNDS (CAROTENOIDS
AND MYCOSPORINES) IN FRESHWATER Patagonian
yeasts
D. Libkind
1
, P. Pérez
1
, R. Sommaruga
2
, M.C. Diéguez
1
, M.
Ferraro
3
, S. Brizzio
1
, H. Zagarese
3
, M.R. Giraudo
1
.
1
CRUB, UNComahue, Bche., Arg.
  2
Institute of Zoology and
Limnology, University of Innsbruck, Austria. 
3
IBB-Intech,
CONICET, Chascomús, Argentina. E-mail: [e-mail]
The synthesis of carotenoids and/or mycosporines (MYC) which
have respectively antioxidant and UVR sunscreen properties is a
common strategy in the photoprotection of microorganisms. The
production of these compounds by freshwater yeast strains grown
under PAR and UVR was assessed. In most red species, caretonoid
synthesis was stimulated under PAR or UVR+PAR irradiation.
This response was higher when constitutive levels of pigments
(in the dark) were lower. This suggests that cells with higher
constitutive levels of carotenoids are less responsive to induction
by UVR-PAR. Four yeast strains were able to produce a UV-
absorbing compound (309-310 nm) when exposed to PAR or UV-
PAR identified as mycosporine-glutaminol-glucoside. This is the
first time that the production of MYCs by yeasts is reported.  All
strains that developed under UVR-PAR were able to synthesize
carotenoids either constitutively or in response to PAR exposure,
and a few of them also produced MYCs. Collectively, our results
suggest that the presence of carotenoids, either alone or in
combination with MYCs, are required for sustaining yeast growth
under exposure to UVR-PAR.
MI-P61.
BIOLOGICAL CHARACTERIZATION OF A
MONOCLONAL ANTIBODY THAT INHIBITS THE trans-
SIALIDASE FROM Trypanosoma cruzi
Pitcovsky Tamara A
1
, Garbarino Gloria
2
, Mocetti Esteban
1
,
Leguizamón M. Susana
2
, and Campetella Oscar
1
.
1
Instituto de Investigaciones Biotecnológicas, Universidad Nacional
de San Martín, San Martín. 
2
Departamento de Microbiología,
Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires,
Argentina. E-mail: [e-mail]
To overcome its inability to synthesize sialic acids de novo,
Trypanosoma cruzi acquires it by transference of the sialyl residues
from the host to parasite glycoconjugates, a reaction catalyzed by
the trans-sialidase (TS). The TS is considered a virulence factor
since it prevents the attack by complement, is involved in the
escape from the phagolysosoma and alters the immune system of
the host. It is also suggested as involved in the invasion of
mammalian cells. No specific inhibitors are already available. After
deleting spurious epitopes from the catalytic region of the TS, a
monoclonal antibody (mAb) collection against the enzyme was
obtained, where only one displayed neutralizing activity (typed as
an IgG2a). This mAb, was tested both in in vivo and in vitro assays.
Differences in infected HeLa cell counts among mAb-treated
cultures and controls were about 25-50%. Passive transfer of mAb
to mice challenged with bloodstream trypomastigotes (RA strain)
failed to significantly reduce levels of parasitemia and mortality.
In contrast, the damage on spleen, thymus and peripheral ganglia
were highly diminished. Our results strongly support the proposed
role of the systemically disseminated TS in the attack of the
immune system.

---