Seminario Biología molecular
Por: Laura Arango Gutiérrez y Santiago Carvalho Saldarriaga
Facultad de Medicina III Semestre- 2014(1)
Universidad Pontificia Bolivariana
Medellín
Similar a Deletion modification enhances anthrax specific immunity and protective efficacy of a hepatitis B core particle-based anthrax epitope vaccine
Similar a Deletion modification enhances anthrax specific immunity and protective efficacy of a hepatitis B core particle-based anthrax epitope vaccine (20)
3. Hepatitis
Is a medical
condition in which
the liver is under an
inflammatory
process.
Normally this
inflammatory
process is due the
action of infectious
agents such as
hepatitis viruses.
Can also be caused
by other infections,
toxic substances,
autoimmune
diseases, trauma and
blood transfusions.
4. Hepatitis
In general, the classic symptom of hepatitis is jaundice caused by
the increase of bilirubin in the blood.
Viral Hepatatis
Hepatitis
Toxic and druginduce hepatitis
Dosedependent
Idiosyncratic
5. Viral hepatitis
Can be caused by
•
•
•
•
•
•
Yellow fever virus
Herpes simplex virus
Cytomegalovirus (CMV)
Epstein-Barr virus
Hepatitis viruses (A,B,C,D,E and G)
Viruses transmitted by transfusion
(TTV)
7. Is an
inflammatory
disease of the
liver caused due
to the infection
with HBV.
The HBV its a
DNA virus who
belongs to the
Hepadnaviridae
family.
8 genotypes of the
virus are known (AH).They have
different distribution
and clinical
presentation.
The hepatitis B
8. Hepatitis B virus
The HBV has
an icosaedric
capsid known
as core
It contains
the viral
DNA and a
polymerase
This complex is
surrounded by
antigens (HBcAg
& HBeAg) and by
an surface
antigen (HBsAg)
9. Hepatitis B virus, genomic structure
Gene • Formation
of the
S
HBsAg
Gene • Polymerase
synthesis
P
Gene • Codifies HBc
and HBe
C
proteins
Gene • Codifies HBx
protein
X
10. Is an infectious disease
caused by the spores of
Bacillus anthracis
Anthrax
The spores can be found
naturally in soil
Commonly affects
domestic and wild
animals (herbivores)
In humans most often
involves the skin, GI tract,
or lungs.
Transmission for get in contact
with infected animals or
contaminated animal products.
11. Anthrax vaccine
The vaccine
is not
typically
available for
the general
public
Anthrax toxin is
composed of a
cell-binding
protein, known
as protective
antigen (PA)
And two
enzyme
components,
called edema
factor (EF) and
lethal factor
(LF).
It's only given to the
people that are in a
high risk of exposure
to the spores
14. General objective
To modify previously created particle HBcL2,
seeking to improve the ability to produce AntiPA and decrease the production of anti-HBc.
16. Construcción del plásmido
PCR inversa: Reacción en cadena de polimerasa- transcripción inversa
• Se utiliza para detección y amplificación de RNA
• Permite estudiar la expresión de determinados genes
Se extrae el RNA total
de las células de
estudio. Se separa
fracción
correspondiente a
RNAm
RNA se transcribe a
DNA mediante una
transcriptasa inversa
Por cada molécula de
RNAm se sintetiza una
molécula de cDNA
monocatenaria
La cDNA monocatenaria
se convierte en
bicatenaria usando una
DNA polimerasa
17. Construcción del plásmido
Secuenciación de DNA
Es un conjunto de métodos
para la determinación del
orden de
los nucleótidos (A, C, G y T)
en un oligonucleótido de
ADN
Verificación de los plásmidos
formados
24. Antígeno protector recombinante y factor
letal recombinante del ántrax
Vector + Fragmento de DNA
DNA recombinante
Replicación del DNA
recombinante dentro de las
células huésped
Aislamiento, secuenciación y
manipulación del fragmento
de DNA purificado
Cualquier molécula de DNA
compuesta de secuencias derivadas
de diferentes fuentes
25. • SDS-PAGE
SDS-PAGE y análisis Western Blott de las
partículas HBc
Migración de una molécula o partícula cargada, bajo la influencia de un campo
eléctrico
26. SDS-PAGE y análisis Western Blott de las
partículas HBc
• Geles de poliacrilamida
• Se puede realizar en
condiciones nativas o
desnaturalizante.
• SDS: Detergente iónico
que
posee
carga
negativa. Considerado
un desnaturante fuerte
27. SDS-PAGE y análisis Western
Blott de las partículas HBc
• Western Blott
Técnica utilizada para detectar
proteínas en un sistema de
reconocimiento basado en la
interacción antígeno-anticuerpo
28. SDS-PAGE y análisis Western Blott de las
partículas HBc
Proteínas se detectan por
inmunodetección: Se usan
anticuerpos monoclonales
previamente conjugados con
compuestos
enzimáticos,
luminiscentes o radioactivos
29. Análisis dinámico de dispersión de
la luz
Es un método óptico
para la determinación
de coeficientes de
difusión de partículas
30. Inmunización animal
HBc144
Grupos de seis
ratones
hembra
H144DL2
H144L2
Inmunización subcutánea
3
Inmunizaciones
cada 2 semanas
Cuarta inmunización: 2
Semanas antes de inyectar
toxina del ántrax
Muestra de sangre después de c/ inmunización
Muestra 6 semanas después de 1 inmunización
Hidróxido de
aluminio= Adjuvante
31. Pruebas serológicas:
ELISA
• Ensayo tipo Sandwich
• Ensayo competitivo
• Ensayo indirecto
Cantidad del producto
formado es directamente
proporcional a la cantidad
de anticuerpo presente en
la muestra
32. Pruebas serológicas:
Neutralización de la toxina in vitro
Se evaluó la capacidad del suero de los ratones
inmunizados para neutralizar la muerte de la línea celular
de macrófagos mediada por las toxinas del ántrax
33. Experimentos de protección del
ratón
Grupo de ratones inmunizados
0, 2, 4, 6, 8, 10, 12 hrs
después del tratamiento
Inyección intravenosa de
LeTx
Monitorizados
2 veces al día por 7 días
41. Discussion
Author
What did he say
Yes/not
Yin et al
We previously sucessfully inserted the
2β2-2β3 loop of PA, which contains a
dominant neutralizing epitope of anthrax,
into the MIR of the HBc
Yes
Petosa et al;
Young and Collier
It seems possible that the insertion of the
foreign sequences in the 78-79 amino acid
regions would increase the total spikes
length outside of the particles
Yes
42. Discussion
Author
What did he say
Yes/not
Billaud et al
Previosly, HBc-based pre immunity was
considered to be one of the system’s
constraints
Yes
Karpenko et al
The assembly was considered a limitation
fro the application HBc particles
No
44. Conclusions
The implementation of virus like particles as a scaffold for the
development of new vaccines and treatments, gives us an
opportunity to fight against a big number of diseases that affect
the world every day, and opens the door for a whole new field
of investigation and scientific advances.
45. Conclusions
The knowledge of the molecular structure of different types of
microorganisms that surround us and the understanding of how
they act gives us an advantage for the design of new techniques
that allow us to fight against them in a efficient and safe
manner, preventing the development of pathologies and
improving the treatment of those who already suffer them.
46. Conclusions
The use of modifiable molecular targets is necessary to find
treatments to many different diseases. It also means that in the
future we can intervene in the apparition of diseases
beforehand playing an important role in preventing them
through the use of vaccines which would significantly better
the patients quality of life
47. Conclusions
In the last few years the perspective on disease and the use of
different techniques has shifted to a more molecular approach,
this will significantly modify the field of medicine because we
will have a more complete approach in the treatment if patients
and the prevention of diseases