plan ti bodies

8/6/2019 Plan Ti Bodies

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Plantibodies Approach

Kabeya J. MuambaDept of Crop PhysiologyUniversity of Agricultural

Sciences, Bengaluru, India


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Antibodies or antibody fragments produced in plants are

often referred to as plantibodies, and they can beexploited for ex planta applications in the field ofmolecular farming.

Many reports describe how very efficiently transgenicplants produce complete antibodies or antibodyfragments, such as Fab and single-chain variable(scFv) fragments


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The structure of antibody


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Single Chain Variable Fragment (scFv) is a fusion ofthe variable regions of the heavy and light chains ofimmunoglobulins, linked together with a short (usuallyserine, glycine) linker.
http://en.wikipedia.org/wiki/Immunoglobulinshttp://en.wikipedia.org/wiki/Serinehttp://en.wikipedia.org/wiki/Glycinehttp://en.wikipedia.org/wiki/Glycinehttp://en.wikipedia.org/wiki/Serinehttp://en.wikipedia.org/wiki/Immunoglobulins


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After isolation and purification from the plant tissue, the antibodies or antibody

fragments can be used in :-

a. ex plantaapplication/ industrial processes, as diagnostic tools, forimmunochromatography, or in medical therapy

b. in plantaapplication , where the strategy is known as

immunomodulation, in which antibodies or antibody fragments areproduced to modulate the function of a corresponding antigen.

As such, immunomodulation can be used to study the function of the antigen or evenof the epitope in plants, to change agronomic traits, or to immunize the plant

against pathogen infection


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Figure 1. In plantaand ex plantaapplications of antibodies and antibody fragments produced in plants. Plants can beengineered to synthesize antibodies in order to obtain pathogen resistance (intra- and extracellular immunization) or tomanipulate the plants metabolism, such as the immunomodulation of enzyme or signal molecule activity. Isolation and

purification, if necessary, allows the use of plant-made antibodies for diagnosis, affinity-based purification, andtherapy.


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In Tobacco plants accumulat scFv fragments in the ER that bind to

abscisic acid (ABA) were shown to wilt under ambient conditions(Artsaenko et al., 1995).

These plants exhibited an increased transpiration rate, indicating that theywere unable to close their stomata.

This might occur because of the concentrated scFv antibodies in the ERprovide an ABA sink and prevent the transport of the hormone and itsinteraction with ABA receptors in the guard cells of the stomata


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Transgenic lines that accumulate high levels of the scFv in the ERshowed a dwarf phenotype and lower gibberellin A1 levels than wildtype

Owen et al. (1992) demonstrated receptor activity modulation with anscFv directed against the plant regulatory receptor protein,

phytochrome.

Seeds from transgenic plants expressing the scFv gene displayedaberrant phytochrome-dependent germination


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INTRA- AND EXTRACELLULAR IMMUNIZATION AGAINSTPLANT-PATHOGEN INFECTION

A major goal of molecular breeding is the generation of transgenic plants thatshow enhanced resistance to pathogen infection

Antibody-mediated virus resistance is seen as an attractive alternative to thevarious forms of pathogen-derived resistance

Most plant viruses are RNA viruses that replicate in the cytosol. As such, thehighest resistance is expected by targeting the antibody to this cellcompartment.


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Besides using immunomodulation to obtain virus resistance, production of ascFv directed against the major membrane protein of the stolbur phytoplasmaproved to be an elegant strategy to control mycoplasma (phytoplasma and

spiroplasma) infection

Plantibody-mediated resistance against complex eukaryotic, multicellularpathogens, such as nematodes, fungi, and insects, remains a majorchallenge.

Alternative methods to control pests, such as the plantibody approach, wouldreduce pollution from synthetic pesticides and make agriculture moresustainable.

Resistance against root-knot nematodes (Baum et al., 1996; Rosso et al.,1996), which is one of the worlds most damaging agricultural pests, has

been tested upon accumulation of the IgM antibody 6D4.

IgM 6D4 binds a glycoprotein of unknown function in the stylet secretions ofthe root-knot nematode, Meloidogyne incognita.


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Isolation of antibody and antibody fragment-encoding sequences

Antibody genes are usually isolated from monoclonal hybridoma cell lines

However, hybridoma technology is expensive and time-consumingand, in general, plant laboratories do not have the facilities to work withanimal cell cultures

Phage display technology, on the other hand, allows the isolation

of antigen-binding scFv-encoding sequences directly from establishedlibraries or from the spleen of immunized mice.

Phage display makes use of generally applied recombinant DNAtechniques


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Monoclonal antibodies (mAb or moAb) are antibodies that are identical because they are produced by onetype of immune cell that are all clones of a single parent cell

Antibody genes are usually isolated from monoclonal hybrid cell lines
http://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Immune_cellhttp://en.wikipedia.org/wiki/Clone_(genetics)http://en.wikipedia.org/wiki/Clone_(genetics)http://en.wikipedia.org/wiki/Immune_cellhttp://en.wikipedia.org/wiki/Antibody


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Phage display is a system in which a protein isdisplayed on the surface of a phage as a fusionwith one of the coat proteins of the virus. TheDNA that encodes this protein is housed within thevirion.

By cloning large numbers of DNA sequences into thephage, phage display libraries are produced with arepertoire of many billions of unique displayedproteins.


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M13 Bacteriophage Structure

The antibody fragment are produced in theperiplasm of E. coli and display on the pIII coat

proteins of M13 bacteriophage

Through several rounds of panning, antigen-binding scFv clones are selected from thelibrary. From the bacterial clones, antibodyfragment-encoding sequences are available for

further cloning in plant expression vectors.


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Gene Function Amino Acids Mol WtII DNA Replication 410 46137

X DNA Replication 111 12672

V Binding ssDNA 87 9682

VIII Major capsid protein 50 5235

III Minor capsid protein 406 42522

VI Minor capsid protein 112 12342

VII Minor capsid protein 33 3599

IX Minor capsid protein 32 3650

I Assembly 348 39502

IV Assembly 405 43476

M13 Bacteriophage Gene Functions


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M13 phage

scFv

scFv isolated from phage display libraries


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With information on the life cycle of the pathogen at hand, a reasonable

prediction can be made on the best way to approach engineeringresistance

The fungi secrete enzymes and toxins essential for fungal pathogenesisas part of an intimate relationship between the host plant and pathogen.These proteins and toxins are suitable targets that could be neutralized

by expressed recombinant antibodies.

It has found that viral infection can be blocked in the cytosol, which isexpected because so many steps of viral reproduction require thecytosol

Conclusion


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The first successful use of antibodies to create a virus resistant plantwas using single-chain antibody fragments specific for artichokemottled crinkle virus (AMCV) (Tavladoraki et al. 1993).

The single chain antibody was constructed from a monoclonalantibody selected from a panel raised against the AMCV virion.

Antibody-based viral resistance is likely to be increased ineffectiveness in the future as antibodies that target viral proteins

crucial for replication (replicase), viral movement, and transmission areused in place of antibodies specific for structural components, likecoat proteins.

Antibody-based resistance will benefit from advances in thetechnology for cytosolic antibody expression.


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WT scFv

Transgenic tomato plants expressing ascFv fragment derived from the F8library are specifically protectedfrom CMV infection


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