molecular characterization and protective inmunity of rhoptry 35(rop35) of toxoplasma gondii as DNA vaccine.

1. Daniel Londoño Cañas
Medicina U.P.B
Tercer semestre 2018-II

2. Introduction
• Toxoplasma gondii, which belongs to the phylum
apicomplexa, is an intracellular parasite that causes
toxoplasmosis disease.
• infects various tissues, including skeletal muscle,
intestine and nervous system forming cysts, that
persist during the life of the host.
• T. gondii infection is usually asymptomatic; however, in
immunosuppressed people it can cause serious complications
and even death, as in the children of women who acquire the
infection during pregnancy.
• Approximately 30% of the world’s population has been
infected by T. gondii because lives an obligate endotrophic
life cycle.

3. • drug-resistant T. gondii has been observed.
• There’s not a effective and safe vaccine, Toxovax® is the
only commercially available vaccine licensed for use in
only sheep to prevent abortions.
• Some of the available drugs against the parasite are
pyrimethamine, sulfadiazine and atovaquone, they
partially control the replication of tachyzoites but do not
prevent the formation of tissue cysts or have action on
existing ones.
• Rhoptry organelle proteins (ROPs) act as a
determinant of secretory pathogenesis playing
important roles in invasion, establishment of the
replicative niche of the parasitophorous vacuole,
host cell manipulation, and resisting host IFN-γ
stimulated innate immunity.
• genes that encode specific ROPs of T.gondii have been
regarded as ideal materials for producing anti-T. gondii
DNA vaccines.

4. Objective
This study aimed to evaluate the protective
effect of a TgROP35 DNA vaccine on
experimental mice subjected to T.gondii
challenge.

5. Materiales y métodos
Ratones y cultivos celulares Parásitos y preparación de antígenos de taquizoitos
solubles (STAg)

6. RT-PCR
Fundamento
• Reacción enzimática que amplifica invitro
un gen o fragmento de DNA o RNA
millones de veces.
• Se usa DNAc a través de la síntesis previa
de RNA por medio de una transcriptasa
inversa,
¿Para que?
• Síntesis de DNAc por medio de la
transcriptasa inversa.
• Para determinar la expresión de
genes como el TGROP35

7. RFLP
fundamento
¿para que?
• reconocer y estudiar la
variación de las moléculas
de DNA intraspecies e
interspecies.
• Enzimas de restricción
fragmentan DNA de forma
especifica.
• Se usa secuencia de DNA
marcada que muestra un
patrón de transferencia
único de un gen especifico
Electroforesis
SDS-PAGE
fundamento
¿para que?
• Separar proteínas y
otras moléculas
según su tamaño.
• Las moléculas se separan en
función de su carga eléctrica,
peso y estructura desplazándose
al electrodo de carga contraria
y a mayor velocidad cuanto
mayor es la carga de la
molécula.

8. ELISA
fundamento
• Se identifica un anticuerpo o
antígeno utilizando un antígeno
absorbido a una superficie.
¿Para que?
• Para determinar los niveles
de anticuerpos en el suero
de los ratones.

9. Resultados
Fig. 1. Agarose gel electrophoresis of the
TgROP35 ORF and identification of
the recombinant plasmid pVAX-TgROP35
digested by KpnI and XhoI. A: (Lane
M) DNA molecular weight marker DL
2000 (ordinate values in bp); (Lane 1)
ORF of TgROP35. B: (Lane M) DNA
molecular weight marker DL 2000
(ordinate values in bp); (Lane 1) the
recombinant plasmid pVAX-TgROP35
digested by KpnI and XhoI; (Lane 2) the
recombinant plasmid pVAX-TgROP35;
and (Lane 3) the pVAXI vector plasmid
digested by KpnI and XhoI.

10. Fig. 3. Phylogenetic tree of the amino acid sequences of TgROP35 from Toxoplasma gondii and
those of ROP35 from other Aplicomplexan species.

11. Fig. 4. Immunoblot analysis of TgROP35 in BHK cells. (Lane M) protein
marker (ordinate values in kDa); (Lane 1) lysates of BHK cells transfected
with pVAXTgROP35 and probed with chicken anti-Toxoplasma gondii sera;
and (Lane 2) lysates of BHK cells transfected with the pVAXI vector
plasmid and probed with chicken anti-T. gondii sera.

12. Fig. 8. Number and size of brain cysts in the mice after challenge
infection with Toxoplasma gondii PRU strain. The number of brain
cysts in the four groups was determined, and the result was
expressed as the mean ± SD. Statistically significant differences
with P < 0.05, P < 0.01 and P < 0.001 are indicated by *, ** and ***,
respectively. The size of brain cysts in each group was observed
with a microscope, and four random fields of vision are presented in
each group. A: number of brain cysts; and B: size of brain cysts (px:
pixel).

13. Discussion
AUTHOR CONTRIBUTION YES OR NOT
Bradley et al.,
Rhoptry proteins play a key role during infection and
are important in
the interaction between the parasite and the host.
Gurunathan et al.
TgROP35 was localized to the parasitophorous
vacuole membrane
(PVM). DNA-based vaccination by eukaryotic
expression vectors, such as pVAXI, has provided an
effective tool for protecting organisms against
pathogen invasion, especially those with parasitic
life cycles in cells. DNA vaccines have notable
advantages because they are easy and
inexpensive to produce and have the capacity to
induce persistent immunity.
Yano et al.
Cope et al.
Cytokines play a significant role in Th cell activation.
IFN-γ, which promotes inflammation, is capable not
only of inducing Th1 cell activation but also of
resisting pathogen invasion.

14. conclusion
Toxoplasmosis is a disease that has become more important in recent
years with the increase in people infected with the human
immunodeficiency virus and those who receive immunosuppressive
therapy for different reasons taking into account that the tests for the
diagnosis of the infection and its interpretation varies depending on the
clinical setting of the disease and It is important to remember that
prevention measures appropriately applied reduce the risks of transmission
of the disease and that it is not the direct contact with the cat that
increases the risk of T. gondii.
it is necessary to continue investigating tgrop35, similar proteins and
potential antigens for the development of a real toxoplasma gondii
vaccine, taking into account that the only vaccine is for sheep and the
gondii resistance against drugs, so priority is needed in develop a DNA
vaccine for human and animal patients for prevention and treatment,
but while this is happening it is important to insist on primary prevention
measures.