2. FOOT-AND-MOUTH DISEASE VIRUS
INTRODUCTION
Foot-and-mouth disease (FMD) is a contagious viral infection common in young children.
Typical clinical symptoms of FMD include fever, as well as a variety degree of blisters and
rotten spots in oral mucosa, breast skin and heels. The virus can exist in the host for a long
time without showing pathological symptom(persitance). There is no specific treatment for
this disease.
Analyzing the immunogenicity of structural protein VP1 of persistent FMDV can provide new
insight to understand the characteristics of persistent infection.
3. E. COLI
E. coli is a bacterium found in the intestines of people and animals, in the environment,
and sometimes also in untreated food and water. Most types of E. coli are harmless
and are part of a healthy intestinal tract.
In this study, the PI-VP1 was cloned into pET-28a vector. PI-VP1 protein was expressed
and purified in E. coli,
INTRODUCTION
4. TO MAKE AN IMMUNOGENICITY
ANALYSIS OF THE E. COLI
EXPRESSED STRUCTURAL PROTEIN
VP1 OF PERSISTENT INFECTION
FOOT-AND-MOUTH DISEASE VIRUS
GENERAL OBJECTIVE
5. PLASMID CONSTRUCTION
Is the construction of rDNA, in which a foreign DNA fragment is inserted into a plasmid
vector. The plasmid is introduced into bacteria through a process called
transformation. Bacteria with the correct plasmid are used to make more plasmid
DNA or gene expression is induced to make protein. In this study it is used to make
more VP1 protein.
METHODS
EXPRESSION AND PURIFICATION OF
THE RECOMBINANT FUSION PROTEINS
Through different chemicals there is an increase in gene expression and therefore of
proteins. Purification consists of eliminating inclusion bodies that are aggregates of
specific types of protein.
6. IMMUNE SERUM ANALYSIS
METHODS
WESTERN BLOTTING
It is a test that is based on the identification of the reaction between an antigen and
an antibody, iFrst, a protein electrophoresis is done and then specific proteins are
identified thanks to an antibody.
This test was done in order to accurately identify the presence and quantity of the VP1
protein.
This test measures the amount of immunoglobulins IgM, IgG, and IgA in your blood. It
was used to observe the immune reaction that the Vp1 protein could cause,
7. En la imagen B realizaron un análisis de material genético del vector pET-28a
que expresa la proteína VP1 por medio de electroforesis. Se puede ver una
banda gruesa de color claro que nos indica la presencia de la proteína.
La imagen C corresponde al análisis de material genético del plásmido
recombinado también por electroforesis. Se observa claramente la franja que
corresponde al material genético que expresa VP1, lo cual evidencia que se
hizo una correcta construcción del plásmido.
RESULTADOS
En este cuadro comparativo podemos ver la expresión de la proteína VP1 en
los antisueron P1-VP1 y PBS . Hay una clara especificidad del P1-VP1 por la
proteína VP1, pero no reacciona cuando hay otro tipo de proteínas libres en el
antisuero. El PBN fue utilizado como control negativo ya que no reacciona a la
proteína diana que se estaba buscando.
8. Se optimizó la concentración de IPTG (Fig. A izquierda), la temperatura de incubación (Fig. A centro) y el tiempo de
incubación (Fig. A derecha). El nivel máximo de expresión de proteína se logró cuando fue inducida por IPTG 1 mM durante 12
h a 37◦C (Fig. A). La proteína recombinante se expresó en un 94,80% de las proteínas totales, principalmente como cuerpo
de inclusión en el sedimento, que requirió un proceso posterior con trituración ultrasónica antes del análisis (Fig. B). La pureza
de la proteína diana podría alcanzar más del 97% después de su purificación (Fig. C) Posteriormente, las muestras obtenidas
se identificaron mediante análisis de transferencia Western con anticuerpo monoclonal anti-His-tag y anti-VP1; se detectó la
proteína diana a partir de la muestra recolectada, lo que demostró que la expresión de PI-VP1 fue exitosa y permitió realizar
más experimentos inmunológicos (Fig. D).
RESULTADOS
9. Author What they said? Did agree?
Hammond et al.
FMD is a highly infectious and contagious disease of
cloven-hoofed animals, which has been considered as
long-term challenge for husbandry in many countries.
According to the serological characteristics, FMD can be
divided into seven serotypes and further divided into many
subtypes
Guangming
Recent years the epidemic scope and outbreak
frequency of type-O FMD have shown a trend of
expansion and enhancement of viral prevalence
Wanxiang
There is no cross immune protection between different
serotypes of FMDV. Besides the genetic variation between
different strains in the same serotype leads to different
antigenicity, hence, limited cross immune protection exists
between different stains in the same serotype.
DISCUSSION
10. 1.
Thanks to molecular biology we can
understand the resistance generated
by viruses such as FMD due to its
protein composition and its ability to
reduce the spread of the virus with a
vaccine, improving human health.
2.
Molecular biology has made great
medical advances in understanding
the different mechanisms used by
pathogens and what are the reasons
that facilitate their proliferation. By
being able to know this, we can deal
with diseases more effectively and
accurately.
CONCLUSION
