Biology Reference
In-Depth Information
Purification of VNPs from infected leaf material is straightforward;
protocols and references can be found at the DPV (http://www.dpvweb.net).
Infected leaves may be homogenized using mortar and pestle or a blender
and the appropriate buffer. The homogenized material is then filtered through
cheesecloth. The virus is purified from other plant material using differential
ultracentrifugation and ultrapelleting. Some protocols include additional
chloroform/butanol extraction and polyethylene glycol precipitation steps.
In any case, the purification protocols are simple, straightforward, and fast.
Using these methods, one person can easily purify up to 200 mg of virus from
100-200 g infected leaf material (typically obtained from around 50 plants)
in a day.
.1.1 Chimeric Virus technology using Plant-Based expression
Systems
One of the advantages of VNPs over any synthetic nanomaterial is the
ability to genetically engineer the particles, in addition to modifying and
functionalizing the particles using chemistry. Genetic engineering allows
one to fine-tune the surface properties of the particles. Amino acids can be
deleted, changed, or introduced. Short peptide sequences can be introduced
in surface loops; even whole proteins can be incorporated into the viral
capsids.
Examples of mutant particles, their applications, and some basic
principles of chimeric virus technology are discussed in Section 3.7.
Genetic engineering of VNPs refers to the manipulation of the genome,
which results in modifications on the protein level. All plant viruses
currently in use for nanotechnology applications have RNA genomes. The
genomes have been sequenced, and the genetic information is available
at the National Center for Biotechnology Information (NCBI) database
(http://www.ncbi.nlm.nih.gov). To perform genetic modifications, a cDNA
copy of the genome is required. The cDNA is the complementary strand of
the genome RNA, which can be synthesized by reverse transcription. The
cDNA can then be amplified as double-stranded DNA using PCR techniques
and inserted into a cloning or expression vector. At this stage, any standard
cloning or mutagenesis procedure can be applied in order to introduce
the desired modification. For detailed background information on cloning
techniques, the reader is referred to textbooks in the fields of molecular
biology and biochemistry.
Once the genetic modification has been introduced into the cDNA clone,
mutant particles can be produced using either a heterologous expression
system (Section 3.2) or a plant-based expression system. Several methods
for the production of infectious mutant VNPs in plants have been developed.
Early methods involved
in vitro
transcription of infectious RNA transcripts,
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