Keywords

1 Introduction

Fasciolosis is a disease caused by the Fasciola genus where Fasciola hepatica and Fasciola gigantica are predominantly found in temperate and tropical climates, respectively. Fasciolosis is traditionally regarded as a disease that affects ruminants which causes large economic losses in the agriculture sector, previously estimated at US$ three billion annually [1], but likely to be far higher currently. In the last 20 years, it has also emerged as an important human zoonosis with an estimated 2.4 million people infected worldwide [2, 3]. In addition, cases of resistance to the drug of choice against fasciolosis, triclabendazole, have been reported in farms of many countries in Europe and in Australia [4–6]. The emergence of triclabendazole-resistant flukes has urged the discoveries of new alternatives to control fasciolosis.

Liver flukes sophisticatedly manipulate the host immune system to maintain their long term survival in the host by shifting the host immune response towards Th2-type, which is anti-inflammatory and promotes wound healing [7–9]. Th1 and Th2-type associated responses in the murine system are reflected by IgG2a and IgG1 isotypes, respectively [10]. The possible requirement of a Th1-type immune response to resist liver fluke infections has been demonstrated in sheep and cattle as IgG2 antibody levels were associated with lower liver flukes recoveries [11, 12]. However, all is not as simple as it seems, as in another study, low levels of IgG2 in sheep were seen as protective [13].

Previously, the efficacy of multivalent vaccines created by combining different cathepsin proteases, DNA vaccines constructed with cathepsin protease encoding genes, and single protein vaccines with various excretory/secretory products as targeted antigens have been evaluated in different studies [14–18]. A protein vaccine with leucine amino peptidase (LAP) as a targeted antigen in alum adjuvant is the only vaccine that fulfilled the requirement to be a commercial vaccine as it appears to have efficacy that has reached the required level and the antigen is delivered in a commercially acceptable adjuvant [14, 19, 20]. Interestingly, the proposed protective pro-inflammatory immune responses induced by this vaccine are low, as indicated by a high IgG1/IgG2 ratio [20]. This would indicate that depending on the antigen, Th2 responses may also be protective.

Parasites secrete various proteases at different stages of their life cycle to facilitate parasitism and maintain their long term survival in the host. In F.hepatica , cathepsin B protease is expressed in the infectious metacercariae and in newly excysted juveniles while cathepsin L isoforms are secreted throughout the life cycle of liver flukes, and more than 80 % of proteins secreted by adult flukes are cathepsin Ls [21, 22]. Therefore, cathepsin proteases play a salient role in F.hepatica parasitism throughout the whole life cycle and could be a potential vaccine candidate, although the cleavage specificity of each cathepsin protease is not fully understood as yet. However, it has been shown that in adult fluke, three cathepsin proteases are secreted being L1, L2, and L5. While they have largely overlapping digestion patterns on most host substrates, L2 can completely cleave collagen, and L5 has a likely specific (but as yet undefined) target [23–26]. This article will focus on using cathepsin proteases as vaccine targets.

In nature, liver flukes produce cathepsin proteases as inactive zymogens initially. Upon secretion into the slightly acidic parasite gut, the acidic environment facilitates the cleavage of the prosegments and produces functionally active matured cathepsin proteases [27, 28]. All secreted cathepsin proteases in liver fluke have multiple disulfide bonds. If recombinant proteins are expressed in either the E.coli or yeast cytoplasm the disulfide bonds will not form, and the enzyme will be inactive [18, 29]. For this reason, the secretory pathway of yeast is used. Recombinant cathepsin protease is secreted by yeast into the culture media in an inactive form and can be activated in vitro by mixing in a buffer with pH range 5–6. The experimental recombinant protein vaccine development steps for fasciolosis are illustrated in Fig. 1.

Fig. 1
figure 1figure 1

Flowchart of experimental recombinant protein vaccine development steps for fasciolosis

Full size image

Yeast expression systems bear the advantages of being unicellular and eukaryotic organisms as they are easy to grow, convenient for genetic manipulation and also with the capability for protein processing, together with the absence of endotoxin and oncogenic or viral DNA. As mentioned above, the expression of eukaryotic protein using prokaryotic system sometimes results in an inactive product due to incorrect folding or certain posttranslational modifications that are missed [30]. Hence, a yeast expression vector is the system of choice to express cathepsin proteases to ensure the activity and the integrity of the recombinant enzymes.

The ability of DNA vaccines to induce both humoral and cellular immune responses that can be manipulated by one of several delivery methods, either to the skin or subcutaneum or muscle, is well known [31]. While the effectiveness of the DNA vaccine in large animals has often been disappointing [32], one of the four licensed DNA vaccines for animal use is against West Nile virus in horses [33]. Furthermore, the route of immunization [29], the species being immunized [32] and the composition of the antigen [34, 35] should be considered as they are also crucial in determining the effectiveness of a vaccine to elicit a protective immune response.

The choice of vectors for DNA vaccines depends on the strategy and the objectives of the study. There are several vectors that were used in previous studies to deliver the antigen to the different cellular types and location in the body of the vaccinated host. Examples are cytoplasmic construct pVR1012, secretory construct pVR1020, chemokine-fused construct pMCP3, lymph node targeting construct pCTLA-4, cytoplasmic pMASIA, and CpG motifs-containing cytoplasmic pBISIA-40 [16, 18, 29]. The experimental DNA vaccine development steps for fasciolosis are illustrated in Fig. 2.

Fig. 2
figure 2figure 2

Flowchart of experimental DNA vaccine development steps for fasciolosis

Full size image

As fasciolosis is predominately a disease of ruminants, it is clear that experimental vaccine studies are preferably undertaken in one of sheep, goats, cattle, or buffalo. The choice of which definitive host is used will largely depend on the parasite species and the predominant region for which the vaccine is a target. Hence, we have previously undertaken vaccine trials in cattle in Indonesia, where F.gigantica is the relevant parasite, and infects large numbers of cattle [36]. In other regions, for example South American countries, sheep are predominately infected and are therefore used as the experimental animals [14].

Having said this, experiments in large animals are expensive, and therefore are often preceded by experiments in commonly used laboratory animals such as mice, rats, and rabbits. While the immune responses to Fasciola in these animals may not mimic those in larger animals, some information regarding the potential effectiveness of vaccine antigen can be gained. As an example, we have used a rat model to test a set of three antigens, together and in combination [37]. The rat model is a perfectly reasonable alternative to ruminants for comparing efficacy between groups.

2 Materials

2.1 Protein Vaccine

2.1.1 Bio-informatics Analysis and Synthesis of Cathepsin Protease Genes

  1. 1.

    Extract nucleotide sequence from GenBank, NCBI.

  2. 2.

    Expasy translation tool.

  3. 3.

    Analyze with codon optimization program.

2.1.2 Cloning of Cathepsin Protease Genes into Expression Vector

  1. 1.

    Synthetic gene in pUC57 cloning vector.

  2. 2.

    YEpFLAG expression vector.

  3. 3.

    S. cerevisiae strain BJ3505 for recombinant protease expression.

  4. 4.

    Restriction enzymes: XhoI, NotI, and 10× buffers.

  5. 5.

    Agarose, loading dye, nucleic acid stain suitable for gel electrophoresis and 1× TAE buffer for running gel electrophoresis.

  6. 6.

    Agarose gel electrophoresis system: for 50 mL of 1.5 % agarose gel, use 0.75 g of ultrapure agarose powder with 50 mL of 1× TAE buffer. Prepare 1 L of 10× TAE stock buffer in Milli-Q water with 48.4 g of Tris base, 3.72 g di-sodium EDTA, adjust to pH 8.5 with glacial acetic acid and dilute to 1× TAE solution prior to use.

  7. 7.

    Gel documentation system.

  8. 8.

    ISOLATE II PCR and Gel Kit (Bioline) for gel extraction.

  9. 9.

    Spectrophotometer for measurement of DNA concentration.

  10. 10.

    T4 DNA ligase and 10× buffer.

  11. 11.

    To make 10 mL ampicillin (100 mg/mL) stock: Add 1 g ampicillin to 10 mL Milli-Q water, sterilize by syringe filter of 0.2 μM pore size.

  12. 12.

    Chemically treated competent cells: E.coliDH5-α. Plasmid pUC57 and YEpFLAG carrying ampicillin resistance marker for positive screening.

  13. 13.

    Heat block/water bath for heat-shock of competent cells.

  14. 14.

    Mg2+ (2 M) stock: Add 2.033 g of MgCl2∙6H2O and 2.465 g of MgSO4∙7H2O into 10 mL of Milli-Q water. Sterilize by syringe filter of 0.2 μM pore size.

  15. 15.

    Glucose (2 M) stock: Add 3.604 g into 10 mL of Milli-Q water and dissolve by swirling. Sterilize by syringe filter of 0.2 μM pore size.

  16. 16.

    SOB media: Add 20 g tryptone, 5 g yeast extract, 0.584 g NaCl, and 0.186 g KCl to 800 mL of distilled water, dissolve the mixture by swirling, adjust pH to 7.0 and top up to 1 L. Sterilize by autoclaving and add 10 mL of 2 M Mg2+ stock.

  17. 17.

    SOC media: Add 99 mL of SOB media with 1 mL of 2 M glucose stock.

  18. 18.

    LB agar supplemented with 100 μg/mL final concentration of ampicillin: Add 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of bacteriological agar into 800 mL of distilled water, dissolve the mixture by swirling and top up to 1 L. Sterilize by autoclaving and add 1 mL of ampicillin stock solution when the temperature has cooled to about 50 °C. Mix and pour into sterile petri dishes. Store refrigerated for up to 3 months.

  19. 19.

    LB broth supplemented with 100 μg/mL final concentration of ampicillin: Add 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl into 800 mL of distilled water, dissolve the mixture by swirling and top up to 1 L. Sterilize by autoclaving and add 1 mL of ampicillin stock solution when cooled. Store up to 3 months at room temperature.

  20. 20.

    Incubator for plate culture.

  21. 21.

    Shaking incubator for broth culture .

2.1.3 Transformation of the Expression Plasmid into Yeast

  1. 1.

    Minimal media supplemented with uracil and lysine, MM + UL (10× solutions): Add 6.7 g of yeast nitrogen base, 20 g of dextrose, 0.02 g of uracil and 0.03 g of lysine to 800 mL of distilled water. Dissolve the mixture by a magnetic stirrer and top up to 1 L with distilled water. Sterilize by syringe filtering using 0.2µM pore size filter. For 1× broth media, add the stock to autoclaved distilled water; for 1× agar plate media, add autoclaved bacteriological agar and mix with the stock solution.

  2. 2.

    Yeast extract peptone dextrose (YPD) media: Add 10 g of yeast extract and 20 g of peptone into 800 mL of distilled water. Dissolve the mixtures by swirling and sterilize by autoclaving. Prepare dextrose separately to prevent caramelization by adding 20 g of dextrose into 200 mL of distilled water. Dissolve the mixture by a magnetic stirrer and sterilize by syringe filtering using 0.2 μM pore size. After the solution has cooled to room temperature, mix both solutions.

  3. 3.

    Bicine solution (1 M): Add 16.3 g of bicine into 80 mL of the Milli-Q water. Dissolve the mixtures by swirling. Adjust the pH to 8.35 and top up to 100 mL. Sterilize by syringe filter of 0.2 μM pore size and store at −20 °C.

  4. 4.

    Sorbitol bicine ethylene glycol (SBEG) buffer: Add 18.22 g of sorbitol, 1 mL of 10 mM bicine (pH 8.35) and 3 mL of ethylene glycol into 80 mL of Milli-Q water. Dissolve the mixtures by swirling and top up to 100 mL. Sterilize by syringe filter of 0.2 μM pore size and store at −20 °C.

  5. 5.

    PEG-bicine solution: Add 40 g of PEG 1000 and 20 mL of 200 mM Bicine (pH 8.35) into 50 mL of Milli-Q water. Dissolve the mixtures by swirling and top up to 100 mL. Sterilize by syringe filter of 0.2 μM pore size and store at −20 °C.

  6. 6.

    NaCl-bicine (NB) buffer: Add 3 mL of 5 M NaCl stock solution and 1 mL of 10 mM bicine (pH 8.35) and make up to 100 mL solution with Milli-Q water. Sterilize by syringe filtering using 0.2 μM pore size and store at −20 °C.

  7. 7.

    Incubator for plate culture.

  8. 8.

    Shaking incubator for broth culture.

2.1.4 Yeast Expression of Cathepsin Proteases

  1. 1.

    20 % dextrose: Add 20 g of dextrose to 100 mL of distilled water. Dissolve the mixtures by swirling and sterilize by autoclaving.

  2. 2.

    60 % glycerol: Add 60 mL of glycerol to 40 mL of distilled water. Sterilize the solution by autoclaving.

  3. 3.

    CaCl2 (1 M): Add 14.7 g of CaCl2∙2H2O to 80 mL of distilled water. Dissolve the mixtures by magnetic stirrer and top up to 100 mL. Sterilize by autoclaving.

  4. 4.

    Yeast expression media (YPHSM): Add 10 g of yeast extract and 80 g of peptone into 700 mL of distilled water and sterilize by autoclaving. Then, add in 50 mL of 20 % dextrose, 50 mL of 60 % glycerol and 20 mL of 1 M CaCl2.

2.1.5 Purification of Cathepsin Proteases

  1. 1.

    Centrifuge to pellet the cells.

  2. 2.

    Nickel-nitrilotriacetic acid chelated sepharose in a column for Immobilized-metal Affinity Chromatography (IMAC): Add 2 mL of nitrilotriacetic acid sepharose into a 5 mL polypropylene column and add Milli-Q water to wash off the ethanol used to suspend the sepharose. Then, add 1 mL of 0.2 M nickel sulfate into the column and wash with Mili-Q water again before equilibrate the sepharose with wash buffer containing 10 mM imidazole prior to use.

  3. 3.

    Tris-glycine SDS-PAGE loading dye, protein marker, Aquastain Stain for SDS-PAGE gel staining and 1× tris-glycine SDS PAGE buffer for running electrophoresis.

  4. 4.

    Tris-glycine SDS-PAGE running buffer (10×): Add 30.22 g of tris, 144.09 g of glycine and 10 g of SDS into 800 mL of Milli-Q water. Dissolve the mixture using a magnetic stirrer and top up to 1 L. Dilute to 1× solution prior to use.

  5. 5.

    NaCl (5 M): Add 146.1 g of NaCl to the 450 mL of Milli-Q water and mix with a magnetic stirrer. Top up to 500 mL.

  6. 6.

    Dialysis buffer (100 mM NaCl): Add 20 mL of NaCl stock (5 M) into 980 mL of Milli-Q water.

  7. 7.

    Imidazole (1 M): Add 34.04 g of imidazole into 450 mL of Mili-Q water and mix with a magnetic stirrer. Adjust to pH 7.6 and top up to 500 mL. Filter through a 0.45 µM pore size syringe filter.

  8. 8.

    NaH2PO4 (1 M): Add 69 g of NaH2PO4.H2O in 450 mL of Mili-Q water. Dissolve the mixture and top up to 500 mL. Sterilize by autoclaving.

  9. 9.

    Wash buffer (25 mM NaH2PO4, 0.5 M NaCl, 10 mM imidazole): Mix 12.5 mL of NaH2PO4 (1 M), 50 µL of NaCl (5 M), 5 mL of imidazole (1 M) and top up to 500 mL with Mili-Q water.

2.1.6 Animals and Vaccination

  1. 1.

    Sprague Dawley male rats at 6 weeks.

  2. 2.

    Quil A (Sigma-Aldrich).

2.2 DNA Vaccine

2.2.1 Bioinformatics Analysis and Synthesis of Cathepsin Protease Genes

  1. 1.

    Extract gene sequence from GenBank, NCBI.

  2. 2.

    Expasy translation tool.

  3. 3.

    Analyze with codon optimization program.

2.2.2 Construction of DNA Vaccines

  1. 1.

    Synthetic gene in pUC57 cloning vector.

  2. 2.

    Cytoplasmic construct pVR1012.

  3. 3.

    Secretory construct pVR1020.

  4. 4.

    Chemokine-fused construct pMCP3.

  5. 5.

    Lymph node targeting construct pCTLA-4.

  6. 6.

    Cytoplasmic pMASIA.

  7. 7.

    CpG motifs-containing cytoplasmic pBISIA-40.

2.2.3 Confirmation of Functional Expression of Antigen by Transfecting COS-7 Cells

  1. 1.

    COS-7 cell line.

  2. 2.

    Lipofectamine reagent.

  3. 3.

    DMEM media.

  4. 4.

    10 % new born calf serum (NCS).

  5. 5.

    6-well sterile tissue culture plates.

  6. 6.

    5 % CO2 humidifier incubator.

  7. 7.

    Amicon ultrafiltration unit.

  8. 8.

    Western blot system.

2.2.4 Animals and Vaccinations

  1. 1.

    Female BALB/c mice.

  2. 2.

    Helios gene Gun System (Bio-Rad Laboratories).

  3. 3.

    NaCl (5 M): Add 146.1 g of NaCl to the 450 mL of Milli-Q water and mix with a magnetic stirrer. Top up to 500 mL.

3 Methods

3.1 Protein Vaccine

3.1.1 Bioinformatics Analysis and Synthesis of Cathepsin Protease Genes ( SeeNotes1–3)

  1. 1.

    Select cathepsin of interest as targeted antigen and download the gene sequence from GenBank.

  2. 2.

    Add appropriate different restriction enzyme recognition fragment (XhoI, NotI) at both ends of the gene sequence to ensure the gene insertion into the expression vector will be in correct orientation.

  3. 3.

    Insert the cathepsin sequence with a stop codon into the insertion site of the pFLAG.

  4. 4.

    Ensure the insert is in frame with the leader sequence encoded by the vector.

  5. 5.

    Insert the cathepsin gene sequence into a codon optimization program and select S.cerevisiaefor codon optimization.

  6. 6.

    Download the codon-optimized sequence and use the sequence for gene synthesis.

3.1.2 Cloning of Cathepsin Proteases Gene into Expression Vector

  1. 1.

    Digest 1 μg of the cloning vector that carries the synthesized cathepsin gene as delivered by the gene synthesized company with the appropriate restriction enzymes (XhoI, NotI).

  2. 2.

    Inactivate the restriction enzyme by heating to 65 °C for 20 min.

  3. 3.

    Load the restriction digested products on a 1.5 % agarose gel and excise the gene fragment.

  4. 4.

    Recover the gene fragment from the agarose gel by using ISOLATE II PCR and Gel Kit.

  5. 5.

    Elute the gene fragment with 30 μL sterile Milli-Q water.

  6. 6.

    Similarly, digest the expression vector YEpFLAG with the same pair of the restriction enzymes and purify the linearized expression vector in the same way.

  7. 7.

    Measure the concentration of both gene fragment and vector.

  8. 8.

    Use the vector to insert molar ratio of 1:5 for ligation.

  9. 9.

    Thaw the chemical treated competent cells, E.colistrain DH5-α on ice for not more than 10 min.

  10. 10.

    Add 1 μL of the ligation product into the cells and keep on ice for 10–15 min.

  11. 11.

    Cell transformation is achieved by heat-shocking the mixture to 42 °C in a heating block for exactly 50 s.

  12. 12.

    Carefully transfer the cells and keep on ice for 2 min without shaking.

  13. 13.

    Aliquot 50 μL of the transformed cells to spread on a LB agar which is supplemented with 100 μg/mL ampicillin.

  14. 14.

    Pick three colonies and subculture in LB broth. Incubate the culture at 200 rpm, 37 °C for 18 h.

  15. 15.

    Isolate the plasmid using the ISOLATE II plasmid mini kit and confirm the insert by restriction enzyme digestion.

3.1.3 Transformation of the Expression Plasmid into Yeast

  1. 1.

    Inoculate a pure culture of yeast strain BJ3505 into 10 mL of YPD media and incubate for 48 h at 30 °C with 160 rpm shaking (seeNote4).

  2. 2.

    Add the 10 mL culture into 100 mL of fresh YPD media and further incubate with the same incubation condition until the absorbance measure at 600 nm reaches about 0.6.

  3. 3.

    Aliquot 10 mL of the culture and centrifuge at 5000 × g for 2 min at room temperature.

  4. 4.

    Discard the supernatant and resuspend the pellet with 5 mL SBEG.

  5. 5.

    Centrifuge the mixture again and resuspend the cells with 200 μL SBEG and incubate at 30 °C with 160 rpm shaking for 5 min.

  6. 6.

    Add 1 μg of the expression plasmid to the cells and incubate at 30 °C for 10 min without shaking.

  7. 7.

    Place the cells at −80 °C freezer for 30 min and then thaw in a 37 °C water bath with gentle agitation.

  8. 8.

    Add 1.5 mL of PEG-Bicine into the cells and mix gently.

  9. 9.

    Incubate the cell mixture at 30 °C for 1 h without shaking.

  10. 10.

    Add 2 mL of NB buffer to the cells and mix by inversion and then centrifuge at 5000 × g for 5 min.

  11. 11.

    Discard the supernatant and resuspend with 500 μL NB buffer.

  12. 12.

    Aliquot 50 μL of the cells and spread on the MM + UL agar plate.

  13. 13.

    Incubate the plate at 30 °C for 7 days.

3.1.4 Yeast Expression of Cathepsin Proteases ( SeeNote5)

  1. 1.

    Inoculate a positive clone into 200 mL MM + UL and incubate for 72 h with 160 rpm shaking at 28 °C.

  2. 2.

    Pellet the yeast cells at 5000 × g for 10 min and inoculate into 2 L of YPHSM.

  3. 3.

    Incubate the cells in YPHSM by shaking at 160 rpm for 72 h at 28 °C.

3.1.5 Purification of Cathepsin Proteases

  1. 1.

    Collect the cells at 10,000 × g centrifugation for 10 min and keep the supernatant.

  2. 2.

    Dialyze the supernatant in dialysis buffer for four times (5 L of dialysis buffer for 1 L of supernatant) in a cold room, stirring with magnetic stirrer, at least 4 h apart between each dialysate change (seeNotes6 and 7).

  3. 3.

    Make up the supernatant to 25 mM NaH2PO4, 0.5 M NaCl and 10 mM imidazole at pH 7.6.

  4. 4.

    Centrifuge the dialysed supernatant at 15, 000 × g for 10 min to remove any precipitate.

  5. 5.

    Use syringe to filter (0.45 μM pore size) dialyzed sample to further remove any particulate matter.

  6. 6.

    Capture the 6 x histidine tagged protein using nitrilotriacetic acid (NTA) resin pre-charged with nickel in a chromatography column.

  7. 7.

    Wash the cathepsin proteases with wash buffer containing 50 and 100 mM imidazole and finally elute with elution buffer containing 250 mM imidazole.

  8. 8.

    To analyze the sample on SDS-PAGE, add 5 μL of 3× SDS-PAGE loading buffer to 20 μL of the eluted fractions and boil the mixture at 100 °C for 5 min.

  9. 9.

    Allow the temperature of the sample to cool to room temperature and load into a 10 % tris-glycine SDS PAGE gel, run electrophoresis at 60 V for the first 30 min and at 180 V for the next 50 min.

  10. 10.

    Visualize the cathepsin proteases by staining with Aquastain solution.

3.1.6 Formulation of Vaccine

  1. 1.

    Add 20 μg of recombinant cathepsin protease in 200 μL 0.9 % saline with 1 mg/mL Quil A.

3.2 DNA Vaccine

3.2.1 Bioinformatics Analysis and Synthesis of Cathepsin Protease Genes (SeeNote8)

  1. 1.

    Select cathepsin of interest as targeted antigen and download the gene sequence from GenBank.

  2. 2.

    Add appropriate different restriction enzyme recognition fragment at both ends of the gene sequence to ensure the gene insertion into the expression vector will be in correct orientation.

  3. 3.

    Insert the cathepsin sequence into the insertion site of the DNA vector.

  4. 4.

    Ensure the inserts are in frame and use the sequence for gene synthesis (seeNote9).

3.2.2 Cloning of Cathepsin Proteases Gene into DNA Vaccine Vector

  1. 1.

    Digest 1 μg of the cloning vector that carries the synthesized cathepsin gene as delivered by the gene synthesized company with the appropriate restriction enzymes.

  2. 2.

    Inactivate the restriction enzyme by heating to 65 °C for 20 min.

  3. 3.

    Load the restriction digested products on a 1.5 % agarose gel and excised the gene fragment.

  4. 4.

    Recover the gene fragment from the agarose gel by using ISOLATE II PCR and Gel Kit.

  5. 5.

    Elute the gene fragment with 30 μL sterile Milli-Q water.

  6. 6.

    Similarly, digest the DNA vaccine vector with the same pair of the restriction enzymes used to digest the cathepsin proteases gene and then gel purify of linearized vector.

  7. 7.

    Measure the concentration of both gene fragment and vector.

  8. 8.

    Use the vector to insert molar ratio of 1:5 for ligation.

  9. 9.

    Thaw the chemical treated competent cells, E.colistrain DH5-α on ice for not more than 10 min.

  10. 10.

    Add in 1 μL of the ligation product into the cells and keep on ice for 10–15 min.

  11. 11.

    Cell transformation is achieved by heat-shocking the mixture to 42 °C in a heating block for exactly 50 s.

  12. 12.

    Carefully transfer the cells and keep on ice for 2 min without shaking.

  13. 13.

    Aliquot 50 μL of the transformed cells to spread on a LB agar which has supplemented with 100 μg/mL ampicillin.

  14. 14.

    Pick three colonies and subculture in LB broth. Incubate the culture at 200 rpm, 37 °C for 18 h.

  15. 15.

    Isolate the plasmid using the ISOLATE II plasmid mini kit and confirm the insert by enzyme digestion and sequencing.

  16. 16.

    Keep the sequence verified construct in DH5-α at −80 °C with 20 % glycerol .

3.2.3 Purification of Plasmid DNA for Vaccination

  1. 1.

    Subculture the sequence verified construct in LB broth for overnight at 37 °C with 200 rpm shaking.

  2. 2.

    Purify the construct using an endotoxin free plasmid Giga kit.

  3. 3.

    Dilute the purified DNA in endotoxin free 0.9 % saline solution at a concentration of 1 mg/mL.

3.2.4 Confirmation of Functional Expression of Antigen by Transfecting COS-7 Cells

  1. 1.

    Grow the COS-7 cells in DMEM medium supplemented with 10 % new born calf serum (NCS).

  2. 2.

    One the day before transfection, seed the cells in 6-well sterile tissue plates in 2 mL complete medium and incubate in a 5 % CO2 humidifier at 37 °C until the cells attain 80 % confluency.

  3. 3.

    Add 4 μg of plasmid to 100 μL of DMEM media without newborn calf serum (NCS) 20 μL of lipofectamine LTX reagent and incubate for 5 min at room temperature.

  4. 4.

    Add the mixture to COS-7 cells and further incubate for 24 h at 37 °C.

  5. 5.

    Add 1 mL of the complex DMEM media with NCS (10 %) to the cells and further incubate for 48 h at 37 °C.

  6. 6.

    Wash the cells with PBS and growth media without NCS and incubate further for 24 h.

  7. 7.

    Harvest the cells and concentrate the supernatant using an Amicon ultrafiltration unit (cut off value 15 kDa).

  8. 8.

    Analyze the concentrated supernatant by SDS-PAGE and western blotting .

3.2.5 Mice and Vaccinations

  1. 1.

    For intramuscular vaccination, prepare 50 μL of 1 mg/mL DNA in 0.9 % NaCl.

  2. 2.

    Inject the vaccine into the midpoint of each thigh muscle.

  3. 3.

    For intradermal vaccination, precipitate 100 μg of plasmid DNA onto 50 mg gold microcarriers (average diameter 1.6 μm) and then use the particles to coat tubing.

  4. 4.

    Cut the tubing into cartridges such that vaccination with a single cartridge will deliver 1 μg of plasmid DNA.

  5. 5.

    Clip and shave the abdominal region of the mouse prior to vaccination.

  6. 6.

    Deliver the particles by a pulse of helium gas at 400 psi.

4 Notes

  1. 1.

    The recombinant cathepsin proteases in the yeast expression vector YEpFLAG will be directed to be secreted by the alpha factor pre-pro leader. The protein sequence labeled with 6× His-tag at the C-terminus will facilitate the downstream purification process. Extension of the cathepsin proteases sequence at the N-terminus with a spacer sequence greatly increased the Kex2p catalytic efficiency which is essential in facilitating efficient cleavage of the alpha factor leader sequence and minimizing hyperglycosylation [38]. Absence of the spacer sequence would cause the retention of recombinant cathepsin in the endoplasmic reticulum [39]. The annotated pFLAG map and the arrangement of the recombinant cathepsin gene are shown in Figs. 3 and 4, respectively.

    Fig. 3
    figure 3figure 3

    Diagrammatic representation of pFLAG constructs. The genes encoding cathepsin proteases are inserted within the multiple cloning sites with various options of restriction enzyme recognition site for gene insertion. Other features of YEpFLAG are: AmpR and TRP1, which allow for selection in E.coliand S.cerevisiae, respectively; CYC1 transcription termination and; the 2μ ori which allows for the replication of the plasmid in S. cerevisiae

    Full size image
    Fig. 4
    figure 4figure 4

    Diagrammatic representation of the cathepsin proteases gene component encodes by pFLAG. The constructs encode the α-factor signal sequence, FLAG epitope/spacer sequence, pro and mature regions of the cathepsin proteases gene and a C-terminus polyhistidine tag. The α-factor signal sequence will be cleaved off upon secretion into the culture media and the FLAG epitope will be removed together with the pro region while activation of the enzyme

    Full size image
  2. 2.

    The N-terminus spacer sequence was not expected to affect enzyme specificity as the pro region is cleaved during activation of the mature enzyme.

  3. 3.

    Stop codon needs to be added to the gene sequence as the YEpFLAG vector lacks a translational stop signal. The YEpFLAG vector uses the ADH2 gene promoter to regulate transcription, where the promoter is tightly repressed in the presence of dextrose. The yeast cells are therefore grown in the presence of dextrose which will be consumed during yeast metabolism, eventually reaching a level at which it is no longer repressing the ADH2 promoter and the recombinant protein will be produced and secreted into the extracellular space.

  4. 4.

    Yeast (S.cerevisiae) strain BJ3505 is carrying mutant uracil, lysine, and tryptophan genes which would be utilized in the selection of positive transformants as the expression vector, pFLAG, contains a complementary tryptophan encoding gene. In addition, strain BJ3505 is protease deficient which will minimize recombinant protease degradation.

  5. 5.

    In general, the passage of the recombinant proteins through the secretory pathway allows posttranslational event such as proteolytic maturation, glycosylation, and most importantly disulfide bond formation. The secretion of the recombinant proteins into the culture media could avoid toxicity from accumulated material in the cells and simplify protein purification process as yeast secretes relatively low levels of native proteins.

  6. 6.

    Prepare dialysis tubing membrane according to the manufacturer’s instruction, where some of the tubing is required to be boiled prior to use. Make sure to choose the correct molecular weight cutoff value of the membrane pore size which should be considerably smaller than the targeted proteins.

  7. 7.

    The length of the dialysis tubing should be longer than just enough to fill all the sample as the water molecules from the dialysate will diffuse into the tubing via osmosis faster than the buffer salts within the sample could diffuse out due to the high salt concentration in the expression media. Osmosis will cause the swelling of the tubing. To concentrate the sample, using polyethylene glycol compound as the dialysate is the method of choice. The molecular size of the polyethylene glycol compound should be larger than the pore size of the tubing membrane to avoid contamination.

  8. 8.

    While DNA vaccines are capable of transfecting professional antigen presenting cells, and can stimulate both humoral and cellular immunity, immune responses elicited are far less than those induced by protein vaccines [40]. The strategies applied to increase the protective immune responses elicited by DNA vaccines are use of secretory vectors, cytoplasmic vectors, use of chemokines for targeting antigen to antigen presenting cells via chemokine receptors [18], and incorporating CpG motifs into the plasmid DNA backbone as unmethylated CpG motif has been found to be a ligand for Toll-like receptor 9 (TLR9) [41].

  9. 9.

    During bioinformatics analysis of the construct for both protein expression and DNA vaccine, it is important to ensure the inserted gene will be translated in frame.