2. Antibody:
Humoral arm of adaptive immunity in
vertebrates
Antibodies, also called immunoglobulins, are a
group of complex glycoproteins produced by B-
lymphocytes.
It specifically recognize and bind to target antigens
on pathogens and leads to its destruction by
complement or ADCC
Immunoglobins have been divided into 5 major
classes on the basis of their physical, chemical, and
immunological properties: IgG, IgA, IgD, IgE and
IgM. They are composed of 2 heavy chains and 2
light chains.
3. PLANTIBODY
A plantibody is an antibody produced by
genetically modified plants
Around 1990, plants were first considered
as a potential host for producing
antibodies and the word “plantibody”
was coined.
Plants are being used as antibody factories
(bioreactors), utilizing their
endomembrane and secretory systems
to produce large amounts of clinically
viable proteins which can later be purified
from the plant tissue.
4. Choice of expression host
The choice of system depends on- storage and downstream processing
1.Leafy crops such as (tobacco- taken as a model plant)
Advantage
Large amount of biomass
Tobacco is a non-food/non-feed crop
Disadvantage
High content of toxic alkaloids and phenolic metabolites, which must be
removed from the final product using additional purification steps.
2. Seed crops
Cereals and legumes produce less biomass than leafy crops but provide excellent
storage properties and leads to easy purification of the end product
Nicotiana tabacum
CEREALS
5. Advantage of using plant over mammal
for the production antibody
Mammalian produced antibody
Could have pathogens deleterious to
humans
Recognized by the human immune
system as foreign and trigger a
rejection response.
Expensive in terms of equipment,
media and the need for skilled
personnel, and there is limited
opportunity for scale up
Plant produced antibody
Less likely to introduce adventitious
human or animal pathogens
They do not trigger immune responses
Lower manufacturing costs
6. To develop a transgenic plant which
produce full secretory antibody against
dental caries produced by Streprococcus
mutans
Streprococcus mutansDental caries
gram-positive coccus
7. Dental caries
Tooth decay, also known as dental caries or cavities, is a breakdown
of teeth due to acids made by bacteria.
Complications may include inflammation of the tissue around the tooth
and tooth loss.
The most common bacteria associated with dental cavities are the mutans
streptococci, most prominently Streptococcus mutans and Streptococcus
sobrinus, and lactobacilli.
Streptococcus mutans are gram-positive bacteria which constitute biofilms on
the surface of teeth.
8. Why secretory IgA antibody against Dental
caries?
Secretory IgAs is the predominant ab that protects against microbial infections at
mucosal sites.
More resistant to proteolysis
Bind Ag with higher efficiency
sIgA applied to the teeth and it is effective at preventing recolonization by S.
mutans for upto 4 months
Tooth decay due to
dental caries by
S.mutans
9. Why in seeds?
Transgenic seeds assure excellent storage properties
Added flexibility in processing management and batch production
Limited range of endogenous proteins in seeds separation of plantibody is less
complicated
11. Monoclonal antibody generated by hybridoma
technology are then used to get the genes
By using copy nature
strategy we can get
gene of 4 different
component of sIgA which
we will insert in
different plants.
Partial sequencing of the
mab for isolating the
corresponding gene.
12. For the construct-
Source of gene- Mouse challenged by S.mutans bacteria
Isolation of this gene-Myeloma B cells by hybridoma technology
Host plant-Engineered mustard plant family (Brassicaceae) in which xylose
transferase is silenced by host engineering
Vector used-pGREEN 4632bp
Express this gene in- Nucleus
Transformation protocol -Agrobacterium mediated nuclear
transformation
Promoter -Seed specific for easy purification Phytohaemoglutinin (PHA –L)
Selectable marker-Mannose-6-phosphate isomerase (pmi)
Mode of action of selectable marker- Alternative C source
Selective agent of selectable marker- Mannose
Reporter gene- Green fluorescent protein (GFP)
Visualise assay by- Non destructive assay, detection by fluorescence
C ter KDEL sequence is added for the retreivel of plantibody from golgi body after
glycosylation
13. Why retaining of ab chain in ER lumen ?
For efficient chain assembly and stability.
Condition in the lumen of the ER favors the correct processing of the
antibody chains especially disulphide bridge formation.
Molecular chaperons help to ab chain folding.
14. pGREEN
4632bp
T- DNA
CONSTRUCT FOR
KAPPA LIGHT CHAIN
Construct
transferred in
agrobacterium
by making the
cell
competent to
uptake
construct
Agrobacterium
successfully uptaken
the construct were
screened by selectable
agent Kanamycin and
taken further to infect
the host plant
15. Glycosylation
• Glycosylation is a enzymatic process that attaches glycans to proteins
• It occurs in all higher eukaryotes in the Golgi complex
• Glycosylation pattern between plant and mammalian expression seem to be similar
with some differences
• Plant has xylose while human don’t have xylose so by host engineering we will
silence xylose transferase enzyme in the host plant.
Human
16. Leaf disc removed from mustard leaf
and incubated with genetically
engineered A. tumefaciens then by
illegitimate recombination T-DNA
inserted into the plant nucleus
Engineered host
plant- mustard
Induced callus
formation by
intermediate ratio of
auxin to cytokinin ratio
Assay for gfp and part
indicated by arrow are
taken for shooting
Shoot formation by
low auxin to
cytokinin ratio
Root formation by
high auxin to
cytokinin ratio
acetosyringone
Transformation process and regeneration of plant
17. Hardening process of transgenic plant- to acclimatize the tissue culture raised plants
Transgenic mustard
growing in tissue
culture lab under
controlled
environment
Plantlets taken for hardening
process from lab
Plantlets taken to
green house
Transgenic plant which are
grown in tissue culture now
growing in green house
After spending some time
in green house these plants
are taken to fields
Plantlet after rooting
and shooting
18. Generation of 4 transgenic plant having
different component of sIgA
After hardening transgenic mustard plant contaning k chain (light chain of
immunoglobin) in the seeds and this transgenic plant is crossed with 3 other
transgenic plants having different components of sIgA .
These 3 other transgenic plant is created having same construct but with
different genes of interest
i. B Lymphoma alpha chain gene (heavy chain of immunoglobin).
ii. B lymphoma J chain gene
iii. B lymphoma secretory component gene
19. Transgenic plant
containing k chain
Transgenic plant
containing alpha
chain
x
Hybrid transgenic plant
expressing both heavy and
light chain whole IgA
x
Transgenic plant
containing J chain
Hybrid transgenic plant
expressing dimeric Ig A
x Transgenic plant containing
secretory component
Hybrid transgenic plant having
secretory IgA
Synthesis of secretory
IgA molecule in
transgenic plant
20. GFP which is used as a reporter marker is also used for screening of
seedsbecause it gives fluorescence
Southern blot analysis
Screening and evaluation technique for plantibody
L NT T
21. Northern blot analysis
Western blot analysis
NT T
L T NT
NT T
22. Extraction of plantibody
Our whole secretory IgA is present in the seeds of hybrid mustard plant after 3 crosses from
where plantibody is generated by downstream processing-
Isolation and purification of endoplasmic reticulum (ER) from seeds by density gradient
centrifugation.
Isopycnic sucrose gradient centrifugation of organelles first purified by molecular sieve
chromatography on Sepharose 4B, however, results in separation of the organelles based on
their differing buoyant densities.
Endoplasmic reticulum isolated by isopycnic density gradient centrifugation can be further
purified by rate-zonal centrifugation
23. Commercially available plantibody
Development
al stage
Phase II in
the USA
Phase II
Preclinical
stage
Phase I
completed
Phase II
completed
Application Plant expression
system
company Product
anti-
Streptococcus
mutans secretory
immunoglobulin
Transgenic
tobacco
Planet
biotechnology
CaroRx®.
Against colorectal
cancer
Transgenic
tobacco
Monsanto NeoRx
Antibody Drug
induced alopecia
Transgenic
tobacco
Planet
biotechnology
DoxoRx
Antibody against
cold causing
rhinovirus
Transgenic
tobacco
Planet
biotechnology
RhinoRx
Antibody for
cancer
Transgenic
safflower
Plant form corp herceptin
25. Thank you
REFERENCES
1. PLANTIBODY: AN OVERVIEW
Priya Jain*, Prasoon Pandey, Dheeraj Jain, Pankaj Dwivedi
2.Plantibodies in human and animal health: a review - NCBI - NIH
3.Journey of Immunoglobulin Protein: An Antibody to Plantibody
4.Plant Bioproducts
edited by Guanqun Chen, Randall J. Weselake, Stacy D. Singer
5.Plant biotechnology
By Adrian slater
6.The isolation of endoplasmic reticulum from barley aleurone layers.
Jones RL
