This power point is about using Nanoparticles as adjuvant to prepare DNA Vaccines

1. Lecturer: Hamid Salari
Teacher: Dr. Kalbassi

2.  Several reports have demonstrated the transient gene expression and effectiveness of
DNA vaccination in fish against viral and bacterial infections
 However, most of these studies have relied on intramuscular injection into fish
 This method is obviously impractical and not feasible for field application.
 Hence, it is necessary to develop simple and cost effective delivery systems to deliver
DNA vaccine for mass vaccination in fish farms

3. Administration of DNA vaccines
Intrabuccal administration
and immersion non-viral gene
transfer agents
Cationic lipids
Cationic
polymers
Encapsulating the DNA
into Chitosan

4. Chitosan is a natural biodegradable polysaccharide extracted from crustacean shells.
Non-toxic
Protect DNA from nuclease degradation
Escherichia coli DH5a

5. Preparation of polyclonal antiserum against OMP38 of Vibrio anguillarum
cloned
Expres.
Rec. prot.
r-OMP38
Confirm. Exp.
SDS-page
Purif. r-OMP38

6. Purif. r-OMP38
BALB/C mice
Producing Polyclonal Anti-serum
in Mice against
r-OMP38
Immobilized Affinity
Chromatography
Anti-serum
Collection
Determination of Anti-serum Concentration
ELISA
1:20000
Immunospecification
Naturally infected Fish
Experimentally infected Fish
Infected by V. anguil

7. Construction and preparation of DNA vaccine
V. Anguilarum OMP38 gene coding
Amplification
Sequencing to confirm the products
Cloned
Selecting the pVAOMP38 by Ampicillin Resistance
E.Coli DH5a
Recombinant cloned plasmid: pVAOMP38

8. Confirming the presence of pVAOMP38 in the E.Coli
Restriction analysis of recombinant analysis of plasmid
DNA sequencing Kit
Purification of plasmid
Store
-20C

9. Preparation of Chitosan-DNA (pVAOMP38) nanoparticles
Chitosan
Sodium acetate buffer (25mM)
Sodium hydroxide… pH = 5.5
Chitosan 0.02%
Sterile: 0.2μm
Plasmid DNA (pVAOMP38)
Sodium solphate Sodium solphate
50 mM 50 mM
Equal volume
Vortex
2500 rpm
30 sec.
Preparation of Chitosan-DNA (pVAOMP38) nanoparticles
mixing

10. Stability of plasmid in Chitosan-DNA (pVAOMP38) nanoparticles
Naked DNA
Chitosan-DNA (pVAOMP38) nanoparticles
Incubation with DNAse I = Elimination of Naked DNA
Neutral pH
15 min
37 C
Stop incubation with EDTA (0.1 M, pH= 8)
Chitosanase Elimination of Chitosan
Chitosan-DNA
(pVAOMP38)
nanoparticles
Evaluation of pDNA integrity by:
Electrophoresis

11. In vitro transfection of pVAOMP38 in seabass kidney cell line (SISK)
SISK Culture
L-15: 24h
Transfection with: Chitosan-DNA (pVAOMP38) nanoparticles
Incubation: 8h
p-Formaldhyde: Cell Fixation
Washing cells with PBS Permeablizing cells with Triton X-100
Mounting Anti-fade (DABCO) Adding Conjugated IgG to cells
Electron microscope

12. Vaccination and efficiency evaluation
Group III: Chitosan-PBS
Sea bass feeding
Group I: Chitosan-pVAOMP38
Group II: Chitosan-pcDNA 3.1
21 day
V. anguilarum Challanged

13. Result
SDS PAGE
Immunospecificity of r-OMP38 polyclonal antiserum
Stability of plasmid in Chitosan-DNA (pVAOMP38) nanoparticles

17. DNA construct
A DNA construct is an artificially constructed segment of nucleic acid that is going to
be "transplanted" into a target tissue or cell. It often contains a DNA insert, which
contains the gene sequence encoding a protein of interest, that has been subcloned
into a vector, which contains bacterial resistance genes for growth in bacteria, and
promoters for expression in the organism. A DNA construct may express wildtype
protein, prevent the expression of certain genes by expressing competitors or
inhibitors, or express mutant proteins, such as deletion mutations or missense
mutations. A DNA construct is often used in molecular biology to analyze
macromolecules such as proteins or RNA in more detail.

18. This pcDNA™3.1(+) vector is designed for high-level, constitutive expression in
a variety of mammalian cell lines. It contains a Geneticin® selectable marker
and a forward-orientation multiple cloning site.
Adjuvant:
a substance which enhances the body's immune response to an antigen.
M cells
special epithelial cells associated with Peyer's patches and lymphoid follicles that
actively take up particulate matter from the intestinal contents. They are probably
the portal of entry for bacteria and viruses.

19. Ovalbumin
The biological function of ovalbumin is unclear although it is said to act as a storage
protein. In research, the ovalbumin (of chicken egg) is used as a molecular weight marker
for calibrating electrophoresis gels. It is also used to stimulate allergic reaction in test
subjects. In medicine, it is administered to patients suspected of being poisoned by heavy
metals (e.g. iron).
Caco-2 cells
The human intestinal Caco-2 cell line originally isolated by
J. Fogh (Sloan Kettering Institute, New York, NY) from a human co-lon adenocarcinoma
(Fogh et al., 1977), is still the best available
cell culture model of absorptive small intestinal enterocytes (Delie
and Rubas, 1997; Le Ferrec et al., 2001; Sambruy et al., 2001) and it
is extensively utilized for toxicological and pharmacological stud-ies.
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is
often used for non-viral methods in eukaryotic cells

20. Fluorescein isothiocyanate (FITC)
a fluorochrome dye frequently coupled to antibodies that are used to locate and identify
specific antigens.
PLGA or poly(lactic-co-glycolic acid) is a copolymer which is used in a host
of Food and Drug Administration
Poly(lactic-co-glycolic acid) (PLGA) is one of the most successfully
used biodegradable polymers because its hydrolysis leads to metabo-lite monomers,
lactic acid and glycolic acid ( Fig. 1). Because these two
monomers are endogenous and easily metabolized by the body via
the Krebs cycle, a minimal systemic toxicity is associated with the
use of PLGA for drug delivery or biomaterial applications

21. Outer polyclonal antiserum (OMP)
Proteins isolated from the outer membrane of Gram-negative bacteria.
BALB/c is an albino, laboratory-bred strain of the House Mouse from which a
number of common substrains are derived.
The complete form, Freund's Complete Adjuvant,(CFA or FCA) is composed of
inactivated and dried mycobacteria (usually M. tuberculosis), whereas the incomplete
form (IFA or FIA) lacks the mycobacterial components (hence just the water in oil
emulsion).

23. An expression vector, otherwise known as an expression construct, is
usually a plasmid or virus designed for protein expression in cells. The vector is used to
introduce a specific gene into a target cell, and can commandeer the cell's mechanism
for protein synthesis to produce the protein encoded by the gene
Recombinant DNA (rDNA) molecules are DNA molecules formed by
laboratory methods of genetic recombination (such as molecular cloning) to bring
together genetic material from multiple sources, creating sequences that would not
otherwise be found in biological organisms.
Recombinant clone
Clone containing recombinant DNA molecules.
Ampicillin Resistance
Nonsusceptibility of a microbe to the action of ampicillin, a penicillin
derivative that interferes with cell wall synthesis.

24. Restriction Analysis
Amplified rDNA (Ribosomal DNA) Restriction Analysis is the extension of the
technique of RFLP (restriction fragment length polymorphism) to the gene encoding
the small (16s) ribosomal subunit of bacteria. The technique involves an enzymatic
amplification using primers directed at the conserved regions at the ends of the 16s
gene, followed by digestion using tetracutter Restriction enzymes. The pattern
obtained is said to be representative of the species analysed. Patterns obtained from
several restriction enzymes can be used to phylogenetically characterize cultured
isolates and 16s genes obtained through cloning from community DNA
Naked DNA
Naked DNA is histone-free DNA that is passed from cell to cell during a gene transfer
process called transformation or transfection. In transformation, purified or naked DNA is
taken up by the recipient cell which will give the recipient cell a new characteristic or
phenotype. Transfection differs from transformation since the DNA is not generally

25. Triton X-100 is a commonly used detergent in laboratories.[5] Some applications include
Ingredient in influenza vaccine (Fluzone)[6]
Permeabilizing unfixed (or lightly fixed) eukaryotic cell membranes
Solubilizing membrane proteins in their native state in conjunction with zwitterionic
detergents such as CHAPS
Part of the lysis buffer (usually in a 5% solution in alkaline lysis buffer) in DNA extraction
Triton X-100
Nonionic detergent used in isolating membrane proteins: the detergent replaces the
phospholipids that normally surround such a protein.