Vaccins - HelhaPHL2010-05 - home

onwardhaggardBiotechnology

Dec 12, 2012 (4 years and 11 months ago)

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Projectleader PHL : E. Wirix


Projectleader HELHa:
J. Schmitz


specialization : Biotechnology












Final
report

Genetic engineering of plants for the
production of medicines

Start date

:











Final

date

:


?
-
11
-
20
10










?
-
12
-
2010







Group 5:

Iris Hensen




Emanuel Casagrande

Jan
-
Pieter Ploem



Thibau
l
t Gabriel

Ilse Timmermans



Table of contents

Table of contents

--------------------------------
--------------------------------
--------------------------------
---------

1

Introduction

--------------------------------
--------------------------------
--------------------------------
----------------

3

General: Genetic engineering

--------------------------------
--------------------------------
--------------------------

4

What’s gen
etic engineering?

--------------------------------
--------------------------------
------------------------

4

History
--------------------------------
--------------------------------
--------------------------------
-------------------

4

Techniques

--------------------------------
--------------------------------
--------------------------------
-------------

4

Recombinant DNA
-
mo
lecules

--------------------------------
--------------------------------
------------------

4

Genomic

libraries

--------------------------------
--------------------------------
--------------------------------
--

4

Eukaryotic

cloning and expression systems

--------------------------------
--------------------------------
--

4

Genetic engineering of plants

--------------------------------
--------------------------------
--------------------------

5

Transformation

--------------------------------
--------------------------------
--------------------------------
--------

5

Dicotyledonous plants
--------------------------------
--------------------------------
-----------------------------

5

Transformation

by
A. tumefaciens

--------------------------------
--------------------------------
-----------

5

Other methods

--------------------------------
--------------------------------
--------------------------------
---

6

Monocotyledonous plants

--------------------------------
--------------------------------
------------------------

6

Biolis
tic transformation

--------------------------------
--------------------------------
------------------------

6

Transformation by
A. tumefaciens

--------------------------------
--------------------------------
-----------

6

Modifying plants

--------------------------------
--------------------------------
--------------------------------
------

7

Hepatitis B

--------------------------------
--------------------------------
--------------------------------
-----------------

7

History
--------------------------------
--------------------------------
--------------------------------
-------------------

8

Contagion and pathogenesis

--------------------------------
--------------------------------
------------------------

8

Structure of Hepatitis B

--------------------------------
--------------------------------
------------------------------

8


Vaccins
----------------------------------------------------------------------------------------------
---------------
6





Introduction

From the beginning of
civilizations
, huma
ns

have
use
d plants. These ca
me

before humans and we

are
still not aware of all their
benefits
. Scientists
and
people who are interested in

these

beautiful things
present in

wild life try to
understand how these
plants operate. More and
more,

they discover a large
variety

of species and t
he
different effects of each of
them

but it is still not
finished.

This text tells us

about the
genet
ic engine
ering of
plants and tries

to
praise


the power of nature
.
Humans exploit
the planet

and its

resources

at the
most and we must develop
some

of these one
s
.

Medicine is in constant
evolution

and
it is necessary
to elaborate
drugs with the
most natural products.
The
development in science is
also in progress and we are
able to modify some genet
ic
aspects. Geneticists are well

advanced in the field of
modifying DNA chain.
Every
day t
hey develop new
techniques
and methods to
tra
nsfer genes from

an
organism to another and
we
intend to develop that
subject here
.


“When there are dozens or
hundreds of patents
involved, negociations can
be labyrinthine”

Dr Richard
Jefferson, Cambia. The
plants world is well
diversified but nevertheless
when geneticists tried to
transfer foreign genes they
only used the
Agrobacterium
tumefaciens

and thought it
was t
he only one able to do
that. N
ew research
s of
which resul
ts were
published

in Nature other
bacteria can carry out gene
transfer. The properties of
A.
tumefaciens

allow scientists
to engineer any desired
genes into the bacterial DNA
and then insert them into
plant genomes via circular
DNA molecules called
plasmid
s. These transfers
can introduce changes to
plants like herbicide or
disease resistance, altered
growth, nutritional qualities,
or the ability to produce
drugs and edible vaccines.


Scientists hope the discovery could
benefit agriculture

All over the worl
d, scientists
discover every day new
treatments, new techniques,
new species,

new

environments and so
on. Not
long ago
, the first
organism able to substitute
one of the six basic
chemical elements
w
as
found

in a

lake of

Eastern
California
. This is one

among many other

proof
s
that we make discoveries
every day

in

chemistry
.

Most GM plants contain a
gene for antibiotic
resistance, but there are
fears this could transfer to
bacteria, making them
immune to common drugs.
Environmental groups say
the work do
es not make the
approval of new GM crops
any more likely. Genetically
modifying plants usually has
a rather low success rate


and researchers need to be
able to select plants which
have successfully taken up
the introduced gene from
those that have not.

W
hat’s genetic engineering?
How does the

genetic
reshuffle work ?

When

were

these techniques
developed? What
tests
have
already
been done ? Which
bacteria

hav
e
been used
to
develop genetic
modification? Why these
bacteria and not
all of
them
?...
These are
s
ome

of
the
questions we will try to
answ
er.











General: Genetic engineering
What’s genetic
engineering?

Everyone knows Dolly the
sheep, the first cloned full
-
grown animal in the world. For
a lot of
people, it was the first
acquaintance with genetic
engineering. However, genetic
engineering is not a new
concept, it already exists for a
lot of years.


Genetic engineering refers to all
techniques that artificially move
or transfer genes from one
organism to another, to produce
new or modified organisms.
The target material is the
deoxyribonucleic acid (DNA)
molecule found in all living cells
of organisms, where genetic
information is sto
red.

(J.Craig
Venter
Institute;Renaldo E. and ph
D. 1
-
7)

History

Genetic Engineering first
appeared in 1972, nineteen
years after the discovery of the
DNA structure. It was Paul
Berg, an American scientist,
who produced the first DNA
-
recombinant molecule.
Recombinant DNA is a type of
DNA that is artificially created
by inserting a strand or more of
DNA into a different se
t of DNA.
Later, in 1976 they bred the first
genetic modified mice. 10 years
after the mice, scientist
approves release of the first
genetically engineered crop, a
gene
-
altered tobacco. These
days, genetic modification is
one of the most important
subjects

in the biotechnology.

(E.Wirix;J.CraigVenter Institute)


Techniques

Recombinant DNA
-
molecules

When a DNA
-
molecule consists
of DNA coming from different
sources, we call it a
recombinant DNA
-
molecule.
Genetic modification can be
applied in bacteria, plants and
in a
nimals. We describe the
process in plants later in
chapter 2.

Restriction
-
endonucleases cut
the genomic DNA in little
fragments. This enzymes
recognize DNA
-
sequences from
4 to 8 nucleotides long. So, a
certain DNA
-
molecule will
always be cut by a certai
n
restriction
-
enzyme at the same
way.


After the cutting, we have some
restriction
-
fragments. Each
fragment will be inserted in a
carrier molecule, which
is
called
a vector. A vector can be:

a
plasmid, bacteriophages,
viruses or
little artificial
chromos
omes.

The restriction
-
endonucleases
split the DNA in a certain way
that the fragments have a
single
-
stranded piece at the 3’
or 5’ end. These ends are
called the ‘sticky ends’. Sticky
ends can interact with sticky
ends from other DNA molecules
which are
cut with the same
restriction
-
enzyme. This clutch
can be made permanently by
another enzyme, DNA
-
ligase.

Afterwards, the recombinant
DNA
-
molecule will be inserted
in a living host cell where they
replicate(cloning).

Genomic

libraries

A DNA library consis
ts of all
recombinant DNA
-
molecules
which are obtained by all
restriction
-
fragments from
ligating a specific DNA
-
sample
in vectors. These recombinant
DNA
-
molecules will be
introduced in cells where the
recombinants will replicate.


When a DNA
-
library cont
ains all
the DNA from the genome from
one organism it calls a genomic
library. It’s also possible to
make a DNA
-
library outgoing
from all the mRNA
-
molecules
that are present in a certain
tissue. These are the cDNA
-
libraries.

Eukaryotic

cloning and
expression systems

These days, it’s possible to
develop recombinant DNA
-
molecules which will be
inserted in the genome of
multicellular organisms. When a
zygote transform with the
strange DNA, it will develop in
an organism which contains the
recombinant DNA in some
cells. If we breed with these
organisms we can get
transgenic organisms, which
contains the recombinant DNA
in all his cells.

(E.Wirix.)


Genetic engineering of plants


Genetic e
ngineering is a
technique used to introduce
desired traits into a chosen
organism.
To achieve this
the scientist has to insert a
specific gene which will
encode for a protein that is
responsible for the
expression of a certain
defined trait. This insertion

followed by the expressi
on
of a new feature is called
‘t
ransformation’.


Tran
sformation of plants is
possible because they are
totipotent:

“it means
the
ability to regenerate from a
single cell to a full grown
plant

.

This
means that if
one cell

is adjusted, all the
cells of the plant will have
the new gene.

Some
examples of

new traits are:


herbicide tolerance, drought
tolerance, resistance to
pathogens and insects
. But
it is al
so possible to insert a
gene that

is responsible for
higher nutritio
n values or for
the production of certain
products that can be of
interest.

The gene used to introduce
the traits can be of any
origin. There is only one
condition, the
trait

has to be
compatible with the host
organism.

To be certain that the plant
is a mo
dified organism the
desired transgene will be
accompanied by a
herbicide/antibiotic
resistance inducing gene.
By adding the herbicide or
the antibiotic, to which the
modified plant should be
resistant, to the nutrition
medium the scientist can
check if the

transformation
worked because only the
plants with the right gene
will be able to grow
.

Transformation

To insert this transgene in
the DNA of the plant the
scientist can chose between
a few techniques, one more
favourable than the other.

There is a differ
ence
between the insertion of a
gene in dicotyledonous and
monocotyledonous plants.

Dicotyledonous plants

There are a few techniques
used to insert a gene in the
genome of the plant.

Transformation

by
A.
tumefaciens

There are more and more
evolutions in the world of
plant genetic manipulation,
A. tumefaciens

still is a
major method of choice for
transforming plant cells.
Despite these progresses,
we always work on this
bacteria to a better
understanding of the
mechani
sm of gene transfer
. A lot of important cereals
have now been transformed
using A. tumefaciens
(Newell, 2000).

A. tumefaciens

seems to be
the best discovery to realize
DNA implant into plant
genomes. Bacterial vectors
such as
Eschericia coli

have
already

been used
successfully as vectors in
microbiology (Kikkert
et al.
,
1999) ;

that is now extended
to the world of botany.
These techniques have
been applied on several
plants, such as lettuce
(Curtis, 1995), rice (Hiei,
1997) and tomato (Tzfira
et
al.
, 2002
). This proves that
direct gene transfer methods
are not the only way for
transforming important crop
plants (Newell, 2000). The
transformation by
A.
Tumefaciens
permits
insertion of specific
sequences of DNA into
plants genome. This is a
good reason to p
refer it to
other methods (see below)
although the success rate is
not 100% (Gheysen
et al.
,
1998). There are however
some valid arguments
against the validity of
A.
tumefaciens

mediated
transformation.

Dicotyledonous plants are
those which develop from
two cotyledons in the seed.
They can be recognized by
the branching veins in their
leaves. Dicots of commercial
value include many
horticultural plants such as
petunias, and crops such as
tobacco, tomatoes
, cotton,
soybean and potatoes
.
Tobacco, due to its ease of
transformation, initially
became the workhorse of
plant genetic engineering,
but more recently the
common wall or thale cress,
Arabidopsis thaliana
, has
become very popular. It has
the advantage
of not
requiring tissue culture
during its transformation.
Tomatoes have been
transformed to delay their
ripening, cotton to insect
resistance and herbicide
tolerance, soybeans to
improved oil quality and
herbicide tolerance, and
potatoes to resist viruses
.




Other methods

For many years the only
alternative to
A.
tumefaciens
-
mediated
transformation was the
direct uptake of naked DNA
by plant protoplasts,
achieved by electroporation
or mediated by polyethylene
glycol (PEG). This depends
on the ability of pla
nts to
regenerate from protoplasts,
which varies considerably
between species. For
example, there is many
parameters to be successful
with PEG technique (such
ion concentration, molecular
weight and concentration of
PEG, physical configuration
of nucleic a
cid,…).
Linearized double
-
stranded
plasmid DNA molecules are
expressed and integrated
most efficiently.

Now the most widely used
alternative to transformation
by
A. tumefaciens
is
biolistics. Other (marginals)
techniques exists, including
pollen co
-
cultiv
ation,
microinjection of somatic
embryos and liposome
fusion with protoplasts.

Monocotyledonous
plants

These plants require a
different approach to insert a
gene.


Biolistic transformation


This technique of particle
bombardment, or biolistics,
is the mos
t versatile and
effective way of creating
many transgenic plant
species, including elite
lines.



When this technique is used
an isolated DNA
-
fragment
has to be coated on a metal
particle. Currently gold and
tungsten are often used
metals because of their
inert
nature.


The coated particles are
shot into the cell with a gene
gun, a biolistic device driven
by a gas, Helium for
example.

When the particles pass
through the cell there is a
chance that the new gene
will be introduced in the
genome of the plant.


Transformation by
A.
tumefaciens

This method is used less
frequently but when it can
be used it is the preferred
technique. The problem
here is that some important
mon
ocots, such as: maize,
rice and wheat
are resistant
to
A. tumefaciens

and
cannot be tran
sformed
.
There have been some
efforts to alter the
A.
tumefaciens

to make it able
to infect these monocots.
Until now the greatest

success
were obtained

with
ric
e.

(
J.A.Thompson
)





Gene transfer was successful in the
tobacco plant















































Modifying plants

Arsenic and its compounds
occur naturally in many
places, but high levels
accumulate in the
environment through wood
preservatives,
fertilizers,
coal burning, paints and
other industrial uses. A
carcinogen over long
-
term
exposure, the metalloid
becomes particularly
troublesome when it
contaminates groundwater.

Known for its arsenic
-
proof
powers, the ladder brake
fern (
Pteris vittata)
i
s already
used in some of the
Environmental Protection
Agency’s

phytoremediation
efforts of arsenic
-
laden soil.


But botanists at
Purdue
University
, hope their
research, published in the
journal
Plant Cell
, might
eventually improve clean
-
up
strategies, vi
a genetic
modification

They’ve isolated the gene
(ACR3) that codes for a
membrane protein within the
ferns’ vacuoles. A second
copy of ACR3 allows the
ladder brake fern to safely
transport arsenic from its
roots to its fronds, where
most of the toxin is st
ored.

Rice or not, genetically
modifying organisms might
also be problematic, even if
certain plants

moss,
lycophytes, gymnosperms,
and other ferns

already
have a single copy of the
gene. Flowering species
(from more recent branches
of plant evolution) app
ear
not to have ACR3 at all.

The researchers’ next move
will be to inject ACR3 into
the genome of a flowering
plant, thale cress
(
Arabidopsis)
, to see if it,
too, can become an arsenic
vacuum.

Images: Wikipedia
Commons


Pteris vittata

Arsenic
-
loving
bacteria

The scientific world used to
think that there were six
basic chemical elements

:
carbon, hydrogen, nitrogen,
oxygen, phosphorus and
sulphur. The other ones are
not essential; they are
sometimes noxious.

Searchers have discorvered

a bacteria in a California
lake, the GFAJ
-
1, which is
not only able to survive
arsenic consumption but it
inserts
atom
s

of it

into its
DNA. Felisa Wolfe
-
Simon, a
searcher at the American
Geophysical institute
(USGS) explains that they
have discovered a mi
crobe
which build a part of itself
with arsenic. The
researchers began to grow
the bacteria in a laboratory
on a diet of increasing levels
of arsenic, finding to their
surprise that the microbes
eventually fully took up the
element, even incorporating
it i
nto

the phosphate groups
that cling to the bacteria’s
DNA.

Is that really a new form
of life

?

Such a bacteria is able to
evolve with arsenic, this
would suggest that forms of
life could be possible on
other planets on which life is
thought to be impossibl
e.

But, beware because other
researchers were done
previously but we will not
speak about them. “It is
important to notice that the
finding is the use of arsenic
by the bacteria to build an
organism” said Ariel Anbar.

That ‘extremophile’ bacteria
was alre
ady known but we
did not know that it survived
arsenic, a priori, an
inhospitable environment.

What are the
consequences ?

“This work shows that, if we
force them, the bacterias
can use arsenic in place of
phosphorus. But they do not
use it spontaneously,
naturally” said Frances
Westall, biogeologist at
CNRS of Orleans.






Hepatitis B
Hepatitis B is an infectious
illness ca
used by hepatitis B
virus
which infects the liver of
primates
, including humans,
and causes an inflammation
called hepatitis. Originall
y
known as
"serum hepatitis",

the
disease has caused epidemics
in parts of Asia and Africa, and
it is endemic in China.[2] About
a third of the world's population,
more than 2 billion people,
have been infect
ed with the
hepatitis B virus.

This includes
350 million ch
ronic carriers of
the virus
. There’s no
relationship between Hepatitis
A and C with B.

History

I
n 1885, a number of cases of '
s
erum

hepatitis’ were located in
Bremen (Germany) after
administered the variola

vaccine (that contained human
lymph) to workers. Just in the
years forty and fifty of the
previous century a clear
distinction was made between '
serum

hepatitis' and '
contagious Jaundice' on the
basis of transmission
experiments. In search of
genetic di
fferences, scientists in
1965 found a particular protein
in blood of Aboriginals that they
called ' Australian
-
antigen'. This
proved to be later the hepatitis
B
-

surface antigen (HBsAg).
The introduction of safe
effective vaccines (plasma
-
prepared in 1983
, and vaccine
DNA VACCINEs in 1986,) have
increased the possibilities for
worl
dwide suppression of
hepatitis B

virus (HBV) to
introduce vaccination programs
on child age.

C
ontagion and
pathogenesis

Hepatitis B is spread mainly by
exposure to infected blood or
body secretions. In infected
individuals, the virus can be
found in the blood, semen,
vaginal
fluid
, breast milk, and
saliva.
It
is not spread through
food, water, or by casual
contact.

Hepatiti
s B also may
be spread from infected
mothers to their babies at birth
(so
-
called 'vertical'
transmission)

After entering
,

the
HB
V

is
spread through the blood
through the body
. By
adherence to specific sensors
the virus is incorporated in liver
cell but
doe
sn’t
damages these.
The immunological response of
the
immune
competent host
to
presence of the Hepatitis B
virus,

determine
s the clinical
picture
.
Cells
whic
h contain
virus antigens, humoral
and
cellular processe
s of the
immune system are clea
red. As
a res
ult of a strong immune
response at the acute stage
,
an
acute

hepatitis can
show up
.
If
the

immune response

as
needed

the virus is managed.
When the immune system
doesn’t response as needed,
a
chronic infection can arise.


The incubation period lasts 4
weeks to 6 months (usually 2 to
3 months). The variation
depends on the amount of virus
in the inoculum, the route of
infection and host factors such
as host immunity.

Structure of Hepatitis
B

Ph
oto


http://www.uwcreative.com/por
tfolio.html

( We will add all
references soon )



The virus particle, (virion)
consists of an outer lipid
envelope and an icosahedral
nucleocapsid core composed of
protein. The nucleocapsid
e
ncloses the viral DNA and a
DNA polymerase that has
re
verse transcriptase activity.

The outer envelope contains
embedded proteins which are
involved in viral binding of, and
entry into, susceptible cells.
The virus is one of the smallest
enveloped animal
v
iruses, with
a virion diameter of 42 nm, but
pleomorphic forms exist,
including filamentous and
spherical bodies lacking a core.
These particles are not
infectious and are composed of
the lipid and protein that forms
part of the surface of the virion,
whic
h is called the surface
antigen (HBsAg), and is
produced in excess dur
ing the
life cycle of the virus.

(LCI;
Nettleman M., et al.)













Vaccins

Recently
,

we see more and
more
the appearance of new
vaccines o
n the pharmaceutical
market.

T
he
s
e

new vaccine i
s
genetically modified but what i
s
the point of his changes?


First, the new generation
vaccines, would be cheaper,
safer and more effective.

For
indeed in the case of live
vaccines have the
disadvantage is to ensure that
the virus is suffici
ently
attenuated to not cause
disease but still respond to the
immune system to produce
these antigens.

Another
disadvantage is the possibility
that the vaccine virus can
recombine with other viral
strain.


The improvement of classical
biochemistry, recomb
inant DNA
technology, peptide synthesis,
molecular genetics and protein
purification has laid the
foundations for the
development of new vaccines
that could be more efficient,
cost effective and have fewer
side effects

.


5 methods are possible for the
gen
etic modification of viruses
to obtain new vaccine:


1.

Recombinant DNA cloning of
immunogenic surface protein

2.

The chemical synthesis of
polypeptide vaccines

3.

Construction of recombinant
vaccines with genes from a


customer for foreign surface
proteins.

4.

The genetic engineering of
pathogens nonmutant

5.

Production of monoclonal
antibody epitopes of surface
proteins of infectious agents

6.

Nucleic acid vaccines

Immunity against influenza
lasts one to two years; this is
due to the fact that chan
ges
regularly envelope
protein.

Scientists have studied
several strains of influenza
virus were isolated and regions
that do not vary and are
sufficient to induce an immune
response.

They have
discovered a peptide of 18
amino acids which can be
synthesized

in the
laboratory.

Join this synthetic
peptide to a carrier protein
provides a vaccine that protects
mice against different strains of
influenza virus.


The live vaccine can also be
created through genetic
engineering.

if the gene from
another organism ar
e
int
roduced into the DNA of
vaccine
.

The vaccine can
produce the corresponding
proteins.

Experimental vaccines
to protect animals against
rabies, herpes, hepatitis B and
influenza were produced in this
way.

25 genes can be
introduced into a same strain;
t
here are plans to produce
multiple vaccines to confer



immunity to several diseases
simultaneously.

Few examples

:

°

FMD was the first disease
against which effective vaccine
has

been produced by gene
splicing;

°
Recombinant vaccines have
been produced against
rinderpest virus.

Over the last 6 years, 65

70%
of the 150 biotech drugs in
today’s market were approved,
11

of which reached
blockbuster sales status in
2004.

Such developments are
encouraging for the future

and
this area seems to be the major
determinant for future
investments.


Several biotechnology
-
based
products are still in the
preclinical and clinical phase,
raising the concerns associated
with clinical based failures.

Oncology,central nervous
system di
seases,
cardiovascular autoimmune
diseases, inflammatory
diseases, diabetes,
hormone/enzyme replacement
respiratory and infectious
diseases are the major
therapeutic areas that are likely
to see significant biotech
product

launches in the next 10
years.









Reference List


E.Wirix. "Biotechnologie."
Van Gen tot Populatie, deel 1.

2010.

J.A.Thompson. "Genetic Engineering of Plants."
Diss. 2009.

J.Craig Venter Institute.
Genetics

and Genomics Timeline.
2004.

LCI. "Hepatitis B." Diss. 2008.

Nettleman M., et al.
Hepatitis B.
2010.

Renaldo E. and ph D. "Genetically Modified Organism: It's Implications to Food Safety and Consumers
Protection." Diss. 2000.

Jody Banks and David Salt,
Plant Cell

advance online publication, 2010

La Nasa marie arsenic et vieilles bactéries

Denis SERGENT

BBC News, "
Science and environment",
Arsenic
-
loving bacteria may help in hunt for alien life


by Jason Palmer Science and technology reporter, BBC News

Plant biotech goes open
-
source
, Paul Rincon, BBC News reporter

Scientists hope to ease GM fears
, Richard Black, BBC News website environment correspondent

Genetic engineering
, Michelle Smith, 2002

www.studentsguide.in

S. Mary Zachariah Faculty of Pharmaceutical Chemistry,
A study of Engineering In Biotechnology
Based Pharmaceuticals