Lecturer: Hamid Salari

Teacher: Dr. Kalbassi

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

Administration of DNA vaccines

Intrabuccal administration and immersion non-viral gene

transfer agents

Cationic lipidsCationic

polymers

Encapsulating the DNAinto Chitosan

Chitosan is a natural biodegradable polysaccharide extracted from crustacean shells.

Non-toxic

Protect DNA from nuclease degradation

Escherichia coli DH5a

Preparation of polyclonal antiserum against OMP38 of Vibrio anguillarum

cloned

Expres.

Rec. prot.

r-OMP38

Confirm. Exp.

SDS-page

Purif. r-OMP38

Purif. r-OMP38

Producing Polyclonal Anti-serum in Mice against

r-OMP38

BALB/C mice

Immobilized Affinity Chromatography

Anti-serum Collection

Determination of Anti-serum Concentration

ELISA

1:20000Im

mu

no

specificatio

n

Naturally infected Fish

Experimentally infected Fish

Infected by V. anguil

Construction and preparation of DNA vaccine

V. Anguilarum OMP38 gene coding

Amplification

Sequencing to confirm the products

Cloned

E.Coli DH5a

Recombinant cloned plasmid: pVAOMP38

Selecting the pVAOMP38 by Ampicillin Resistance

Confirming the presence of pVAOMP38 in the E.Coli

Restriction analysis of recombinant analysis of plasmid

DNA sequencing Kit

Purification of plasmid

Store

-20C

Preparation of Chitosan-DNA (pVAOMP38) nanoparticles

ChitosanSodium acetate buffer (25mM)

Sodium hydroxide… pH = 5.5

Chitosan 0.02%

Sterile: 0.2µm

Sodium solphateSodium solphate

Plasmid DNA (pVAOMP38)

50 mM 50 mM

Equal volume

Vortex

2500 rpm

30 sec.

Preparation of Chitosan-DNA (pVAOMP38) nanoparticlesmixing

Stability of plasmid in Chitosan-DNA (pVAOMP38) nanoparticles

Naked DNA

Chitosan-DNA (pVAOMP38) nanoparticles

Incubation with DNAse I = Elimination of Naked DNA

Neutral pH15 min 37 C

Stop incubation with EDTA (0.1 M, pH= 8)

Chitosan-DNA (pVAOMP38)

nanoparticles

Chitosanase Elimination of Chitosan

Evaluation of pDNA integrity by:

Electrophoresis

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

Adding Conjugated IgG to cells Mounting Anti-fade (DABCO)

Electron microscope

Vaccination and efficiency evaluation

Sea bass feeding

Group I: Chitosan-pVAOMP38

Group II: Chitosan-pcDNA 3.1

Group III: Chitosan-PBS

21 day

ChallangedV. anguilarum

ResultSDS PAGE

Immunospecificity of r-OMP38 polyclonal antiserum

Stability of plasmid in Chitosan-DNA (pVAOMP38) nanoparticles

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 subclonedinto 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.

DNA construct

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 cellsspecial 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.

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 byJ. Fogh (Sloan Kettering Institute, New York, NY) from a human co-lon adenocarcinoma(Fogh et al., 1977), is still the best availablecell culture model of absorptive small intestinal enterocytes (Delieand Rubas, 1997; Le Ferrec et al., 2001; Sambruy et al., 2001) and itis 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

a fluorochrome dye frequently coupled to antibodies that are used to locate and identify specific antigens.

Fluorescein isothiocyanate (FITC)

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 twomonomers are endogenous and easily metabolized by the body viathe Krebs cycle, a minimal systemic toxicity is associated with theuse of PLGA for drug delivery or biomaterial applications

Proteins isolated from the outer membrane of Gram-negative bacteria.

Outer polyclonal antiserum (OMP)

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).

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 cloneClone containing recombinant DNA molecules.

Ampicillin ResistanceNonsusceptibility of a microbe to the action of ampicillin, a penicillin derivative that interferes with cell wall synthesis.

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

Restriction Analysis

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

Triton X-100 is a commonly used detergent in laboratories.[5] Some applications includeIngredient in influenza vaccine (Fluzone)[6]

Permeabilizing unfixed (or lightly fixed) eukaryotic cell membranesSolubilizing membrane proteins in their native state in conjunction with zwitterionicdetergents such as CHAPSPart of the lysis buffer (usually in a 5% solution in alkaline lysis buffer) in DNA extraction

Triton X-100Nonionic detergent used in isolating membrane proteins: the detergent replaces the phospholipids that normally surround such a protein.