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1 – Instituto Superior Tecnico, Lisboa; 2 – Escola Superior de Turismo e Tecnologia do Mar, Peniche

EXTENDED ABSTRACT Production, purification and refolding of recombinant OmpK for ELISA assays: testing DNA vaccination against Vibrio alginolyticus in Solea senegalensis fish. Mariana Jerónimo1, Marília Mateus1 & Teresa Baptista2

Vibrio alginolyticus is one of the Vibrio pathogens common to marine animals with economic importance to aquaculture and larviculture industry. As demonstrated that some outer membrane proteins of Vibrio species induce protective immunity against the bacterium. V.alginolyticus OmpK contains a high number of conserved regions in its sequence and high similarity with other Vibrio species’ OmpK. DNA vaccines can induce immunologic responses involving antibodies production. The gene encoding OmpK was thought adequate to construct DNA vaccine against Vibrio species. The plasma of Solea senegalensis fish vaccinated with the plasmid containing the ompK gene were in ELISAs to test if vaccination induced antibody production. To perform ELISAs, the OmpK was used as antigen. First, pET21a-ompK plasmid was constructed to transform E.coli cells to express the gene encoding the r-OmpK. Then, r-OmpK was purified, refolded and characterized by different techniques. Finally, 110 (mgrefolded-protein/gcell-dry-weight were obtained The OmpK protein renatured in 0.1 % lutensol buffer showed the most tertiary structure and was used in immunological tests. The ELISAs were executed in a very preliminary way to optimize the protocol for future assays but the analysis of the results demonstrated that the vaccination with naked DNA did not increase the fish immunity against Vibrios. Keywords: Vibriosis, OmpK, DNA vaccine, inclusion bodies purification, protein refolding, ELISA

INTRODUCTION Vibriosis, caused by bacteria of the genus Vibrio including Vibrio alginolyticus, is one of the most prevalent disease in fresh and salt-water fish and shellfish species (Ningqiu et al., 2010; Frans et al., 2011; Austin & Zhang, 2006). Some of these species have economic importance to the aquaculture and larviculture industry. (Ningqiu et al., 2010). The important factors that can weaken the fish immune system and induce vibriosis include chemical stress like water quality, pollution, diet composition, biological stress like population density, presence of the micro- or macroorganisms or physical stress however, an outbreak of vibriosis only occurs when water temperature exceeds 15° C (Frans et al., 2011; Austin & Zhang, 2006). The mode of fish infection by Vibrio spp. is still in discussion in the literature. Vibriosis in fish usually occurs through the skin penetration but also can occurs through oral intake of the pathogen presenting in water or food (Frans et al., 2011; Austing & Zhang, 2006). These bacteria are responsible for fatal haemorrhagic infection and the typical external clinical signs include weight loss, lethargy, red spots on the ventral and lateral areas of the fish and swollen and dark skin lesions that can ulcerate and bleed. However, in some acute cases, the infection spreads so rapidly that most of the infected fish die without showing any clinical signs (Frans et al., 2011; Actis et al., 1999). Due to the mortality power of the disease a rapid, accurate and reliable detection is crucial to minimize economic losses caused by Vibrios (Frans et al., 2011). Vibriosis is prevented and treated by various strategies such as antibiotics, however antibiotics can be rendered ineffective due to misuse, resulting to the development of resistant strains. Therefore, researchers are developing other methods of prevention and treatment of this disease. (Nehlah et al, 2016). As a Gram-negative marine bacterium, V. alginolyticus’ cell wall structure presents an outer membrane, consisting of protein, lipid and sugar. The Outer Membrane Proteins (OMPs) play an important role in the interaction between the bacteria and the host during infection. Its components are easily recognized as foreign substances by the immune defense system of the hosts. Among other factors, these proteins contribute to V. alginolyticus pathogenicity (Ningqiu et al., 2008; Nehlah et al., 2016).

OmpK is an outer membrane protein found in pathogenic bacteria. The OMPs of Vibrio species are highly immunogenic components due their exposed epitopes on the cell surface (Li et al., 2010). OmpK, as one of the most important OMPs, is widely distributed among Vibrio and Photobacterium and was shown to be the receptor of KVP40, a broad-host-range vibriophage in V. parahaemolyticus (Qian et al, 2008). By polymerase chain reaction (PCR) and sequencing, open reading frame (ORF) of ompK gene obtained from prepared genome DNA of V. alginolyticus strain ZJ04107 presented a full-length of 846 bp. The deduced amino acid sequence of ompK gene consists of 281 amino acid residues with predicted molecular weight of 31.3 kDa. The mature OmpK protein consists of 261 amino acids (GenBank accession no. DQ063588) (Qian et al., 2008). In 2008, Rong-Hua Quien had demonstrated with the help of Blastn in GenBank that, the OmpK of V. alginolyticus presents high similarity with other Vibrio spp., which indicated that OmpK protein was highly conserved. The alignment analysis of the translated amino acid sequences of OmpK also showed that it was highly conserved, which could serve as a surface antigen for good vaccine candidate (Qian et al., 2008). Ningqiu Li also demonstrated that OmpK is good as a versatile Vibriosis vaccine candidate since polyclonal antibody raised against the recombinant OmpK from V. harveyi strain EcGs020802 recognized the OmpK homologues from another Vibrio spp. by immunoblotting (Ningqiu et al., 2010). These studies showed that the conserved OmpK is an effective vaccine candidate against infection by Vibrio spp. as preventions, instead of using antibiotics as treatment.

On the other hand, DNA vaccines, termed “The Third Generation of Vaccines”, involves the direct introduction into living host of a plasmid containing the DNA sequence encoding the antigen(s) triggered an immune response. The recent successful immunization of experimental animals against a range of infectious agents and several tumour models of disease with plasmid DNA testifies to the powerful nature of this revolutionary approach in vaccinology (Koprowski & Weiner, 1998). A major attraction of DNA vaccines over conventional vaccines is that they are able to induce protective cytotoxic T-cell responses as well as helper T-cell and humoral immunity - involving antibodies and is often called antibody-mediated immunity that is the case of the DNA vaccine against Vibrio species (Alarcon et al., 1999). Current vaccine research is oriented to replace conventional vaccines with new more

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effective and safer approaches (Kumar et al., 2007). DNA vaccines provides several potential advantages over other traditional vaccines such as low cost and ease of production in large-scale, ease of manipulation, increased stability, only express antigen of interest, and as said before, generates humoral and cell mediated immune responses (Ghaemi et al., 2007; Li et al., 2012; Alpar et al., 2005). Bacterial plasmids with vaccine inserts are constructed using recombinant DNA technology – biomolecular biology techniques. Then, bacteria are transformed with these bacterial plasmids and bacterial growth produces multiple plasmid copies. The plasmid DNA is then purified from the bacteria using specific protocols that ensure the separation of the circular plasmid from the genomic DNA and other cellular constituents. This purified DNA acts as a vaccine (Schirmbeck et al., 2001). As said before, DNA vaccines are easier to purify than those of protein, and have other good advantages. Only a few studies on DNA vaccines against fish bacterial pathogens have been reported but it was thought that a DNA sequence compressing a plasmid and ompK gene could be a good candidate vaccine against Vibrios. This research focused on production, purification and refolding of recombinant OmpK for ELISA to test DNA vaccination against Vibrio alginolyticus in Solea senegalensis fish with a plasmid containing ompK gene. To produce r-OmpK protein, the construction of the pET21a-ompK plasmid was necessary to transform E. coli cells. The r-OmpK was produced by these transformed cells when induced with IPTG. The pET21a-ompK restriction map was presented in the figure 1.

Figure 1 Restriction map of pET21a-ompK designed in SnapGene. pET21a contains resistance to ampicillin and lac operon.

Once the OmpK is a transmembrane protein and it was overproduced in the cell, it was also expected that forms aggregates nominated as inclusion bodies (IBs). The methods used for protein purification were based in several wash and centrifugation steps since IBs are insoluble and precipitate. After purification, the IBs were denatured and r-OmpK had to be renatured to be used as antigen. To study if r-OmpK took some tertiary structure, UV and fluorescence spectroscopy techniques were performed. When the protein was purified, refolded and lastly analysed, was used to perform the ELISA with plasmas of vaccinated fish with a plasmid containing ompK sequence. METHODS AND MATERIALS Escherichia coli (DH5a) growth to produce pET21a E. coli (DH5a) containing the pET21a-ZZcbm64 used in this experimental work was derived from another research project of iBB. Aliquots of E. coli containing pET21a were withdrawn from cell banks. However, the plasmid also had contained a DNA sequence coupled, ZZ-cbm64 with 630 bp because the plasmid was used in another research. The presence of pET21a gives the cells ampicillin resistance.

Aliquots of E. coli harbouring pET21a were withdrawn from cell bank. However, the plasmid also contained a coupled DNA sequence, ZZ-cbm64 with 630 bp. The presence of pET21a gives the cells ampicillin resistance. To inoculate a culture from the - 80 °C stock, 100 µL of bacterial cells from one cryovial were grown in LB broth (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl; pH = 7.0) that it was prepared according to the manufacturer indications, 25 g LB powder (Sigma) per 1 L distilled water. Then, 5 mL of LB medium was introduced into 15 mL Falcon tubes and autoclaved at 121 °C for 20 minutes. Afterwards, 5 µL of 100 mg/mL ampicillin solution and the inoculum were added to the warm sterilized medium. The growth was performed at 37 °C and 250 rpm and was monitored by OD (optical density) measurement at the wavelength of 600 nm using a HITACH U-200 spectrophotometer. The acquired values were used for the construction of growth curves that allowed the reach the beginning of the stationary phase of cell growth observation. Then E. coli cells were harvested in a refrigerated centrifuge (VWR micro star 17R) at 6000 g for 3 minutes and pET21a- zzcmb64 were purified using High Pure Plasmid Isolation Kit (NZYTech). The concentration of the plasmid solution was measured in a NanoVue plus (General Electric). For vector preparation, two restriction enzymes were used (Xho I and Nde I (ThermoFisher)). The buffer and reaction conditions were those recommended by the manufacturer. pET21a band was cut and the plasmid from gel slice was recovered using plasmid DNA purification from Agarose Gels (NZYTech). The pDNA concentration was measured in the NanoVue (General Electrics).

Insert preparation - ompK gene The ompK gene was obtained by PCR. The DNA template used in PCR was pVAX-ompK previously constructed by Joa ̃o Filipe Paulo. The primers used for DNA amplification were also selected by him. KOD Hot Start was the selected DNA polymerase enzyme and the followed protocol was that suggested by the manufacturer (KOD XtremeTM Hot Start DNA Polymerase | 71975. 2017). The primers that come freeze-dried from the manufacturer were suspend in a given volume to obtain a solution with final concentration of 10 μM. The stock of pVAX-ompK was concentrated at 291 ng/mL. After amplification reaction, the ompK concentration was measured by NanoVue plus and the protocol for PCR clean-up (NZY tech) was used to purify ompK gene. Thereafter, ompK had to be digested with the same restriction enzymes (Xho I and Nde I) used in the digestion of pET21a so that it could be ligated and the pET21a-ompK plasmid constructed. The ompK final concentration was measured by NanoVue plus. pET21a-ompK ligation The ligation reaction was adapted from T4 DNA Ligase Blue/White Cloning Qualified Protocol (2017). For the ligation, T4 ligase was used. The amounts used of pET21a and ompK depend on the size of the vector and the DNA insert (vector size – 5363 bp; insert size – 781 bp). The ligation mix was incubated for 4 h at room temperature. After these 4 h, part of the mixture (5 µL) was used to transfect chemically competent E. coli (DH5a) cells directly and another part was stored overnight at 4 ° C to transfect cells from another bank of chemically competent E. coli (DH5a) cells - also with 5 µL of ligation solution. Transformation of E. coli (DH5a) cells with pET21a-ompK Transformation was performed with the pET21a-ompK prepared immediately after the ligation reaction and after pET21a-ompK was rested overnight at 4 °C. First, 50 ng of pET21a-ompK was added to a cryovial with chemically competent cells from the bank and

pET21a-ompK

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incubated 30 min on ice. Afterwards, the mixture was heated for 1 min at 42 °C and further rested for 2 min on ice. Then, 1 mL LB medium was added to the mixture which incubated for 1 h at 37 °C. Selection of transformants was accomplished by platting on LB agar medium containing 100 µg/ml ampicillin (Roche) and cells incubation for 24h at 37 °C. After these 24h at 37 °C, colonies of transformed E. coli cells had grown, were picked from the Petri plate and inoculated in 15 mL centrifuge tubes containing 5 mL of LB medium with 100 µg/mL ampicillin and cultured until reaching the beginning of the stationary phase. Transformed cell culture was performed at 37 °C until OD=1 was reached in liquid medium and aliquots of these cells were banked at -80°C until use. Analysis of pET21a-ompK recombinants To verify if the transformation with the ligation pET21a-ompK was successful, High Pure Plasmid Isolation Kit (NZYTech) was performed to obtain the plasmids from transformed cells grown in 5 mL LB medium + 100 µL/mL ampicillin until reaching the beginning of the stationary phase. Final concentration of pDNA was measured by NanoVue plus. Then, the plasmids were digested with the action of a restriction enzyme: Eco RV (Thermofisher). The buffer and reaction conditions were those recommended by the manufacturer. After digestion reaction, the mix was run on an 1% agarose gel to check the presence of pET21a-ompK transformation. E. coli BL21(DE3) transformation and verification of the presence of pET21a-ompK The process for E. coli BL21(DE3) transformation and confirmation of the pET21a-ompK presence followed the protocols used for E. coli (DH5a). Expressing the OmpK protein After the pET21a-ompK was establish in E. coli BL21(DE3), expression of the target DNA was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) (Fisher Scientific) to a growing culture. Since the vector had the T7lac, a final concentration of 1 mM IPTG was recommended for full induction. Cells containing pET21a-ompK from a cryovial were inoculated in 250 mL erlenmeyer having 30 mL LB medium supplemented with ampicillin (100 µL/mL) and incubated overnight at 37 °C and 250 rpm. Then, the OD at 600 nm was measured and the amount of cell suspension (necessary for the initial OD600 nm = 0.1) inoculated 1L erlenmeyer having 150 mL of the same medium which incubated under the same growth conditions until OD600 nm had reached 1. Next, IPTG from a 1 M stock solution was added for a final concentration of 1 mM and the incubation continued for 6 h. The final OD600 nm was measured on HITACH U-2000 spectrophotometer. Thereafter, the cells were harvested by centrifugation (Sorvall RC6 – SLA3000 rotor) at 5000 g for 5 min at 4 °C and the supernatant was discarded. The pellets were weighed and stored at -20 °C. The procedures described in this section were also performed with non-transformed E. coli BL21(DE3) cells as a negative control to facilitate the identification of OmpK by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (negative control). Lysing cells To break the cells, 5 mL TST buffer (for a final OD=4) was added to the harvested by centrifugation cells that were subjected to the physical method - sonication. TST buffer consisted in 50 mM Tris (Sigma), 150 mM NaCl (Panreac) and 0.05 % (v/v) Tween 20 (Sigma-Aldrich). Afterwards, cells were resuspended and kept on ice to be sonicated. The sonication was performed for 12 min (30 s pulse, 30 s pause) at 30 W in a probe-type sonicator sonoplus

(Bandelin; Probe MS-72). The 5 ml of sonicated cells were divided into aliquots of 250 µL and stored at -20 °C. r-OmpK verification – SDS-PAGE To verify the OmpK presence in cells, a 250 µL aliquot of lysed cells was centrifuged (VWR micro star 17R) for 30 min, 10,000 g at 4 °C. The pellet and the supernatant were stored separately at 4 °C since it was not known if the protein was soluble or insoluble, in aggregates known as inclusion bodies. This procedure was performed both for the induced transformed E. coli BL21(DE3) cells and for induced non-transformed E. coli BL21(DE3). The study of the presence of r-OmpK in pellet or in supernatant was performed by polyacrylamide gel electrophoresis. The 12 % polyacrylamide gel electrophoresis was performed for 2 hours at 90 V for both protein samples.

Purifying r-OmpK protein Three protein purification methods were chosen considering that the protein was aggregated forming IB. The study of the purification methods was followed by running the samples of the supernatants and the pellets into polyacrylamide gels. Method 1 - After sonication, 350 µL of 1% SDS solution was added to a 350 µL aliquot of cell lysate and incubated for 30 min at 4 °C. Then, 3 µL DNase I (Promega) and 3 µL of 1 M MgSO4.7H2O (Sigma-Aldrich) were added to the mixture and incubated for 30 min at 37 °C. The protein aggregates were separated by centrifugation for 15 min at 12,000 g and 4 °C. The supernatant was discarded but stored at -20 °C for further studies. IBs were washed once with 500 µL wash buffer (50 mM Tris-HCl (Sigma- Aldrich), 1 mM ethylenediamine tetraacetic acid (EDTA) (Panreac), 100 mM NaCl and 0.5 % (w/v) Triton X-100 (Merck)) and centrifuged at 12,000 g for 15 min at 4 ° C. The supernatant was discarded again. The pellet was further washed with PBS and re-centrifuged for 15 min at 12,000 g and 4 °C. The washed pellet was stored at -20 °C. The protocol was adapted from Carrio et al., 2000. Method 2 - After sonication, 350 µL cell lysate suspension was centrifuged for 1 h at 17,000 g and 4 °C. The supernatant was discarded and the pellet resuspended in 500 µL wash buffer containing 100 mM Tris-HCl (Sigma-Aldrich) pH7, 5 mM EDTA (Panreac), 5 mM DTT (Sigma-Aldrich), 2 M urea (Sigma-Aldrich) and 2 % (w/v) Triton X-100 (Merck). The suspension was centrifuged for 30 min at 17,000 g and 4 °C. The supernatant was discarded. This last step was repeated three times. The pellet was then washed with 500 µL wash buffer without the Urea and Triton X-100. Then, it was centrifuged for 30 min at 17,000 g and 4 °C. The two steps mentioned before were performed one more time. Washed pellet was stored at -20 °C. The protocol was adapted from Palmer & Wingfield, 2004. Method 3 - After sonication, the 350 µL cell lysate suspension was centrifuged for 10 min at 10,000 g and 4 °C. Then, the supernatant was discarded and the pellet was added to 500 µL lysis buffer (50 mM NaCl (Panreac), 1 mM EDTA (Panreac) and 0.5 % (w/v) Triton X-100 (Merck)). After that, the mixture was incubated for 5 minutes at room temperature and centrifuged for 15 min at 12,000 g at 4 °C. The last steps were performed one more time. The washed pellet was stored at -20 °C. The protocol was adapted from Valente et al., 2006. Quantification of total protein – BCA Protein assay For the quantification of the protein it was used Pierce™ BCA Protein Assay Kit, a colorimetric test. The absorbance was measured in the SPECTRA max plus (Molecular devices) at 562 nm.

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Once the protein was presented in IBs, denaturation of r-OmpK before refolding process was necessary. Refolding of the protein was performed using consecutive dialyses by changing only the contents their concentrations of the buffers. The protocol was adapted from Rogl et al., 2008. Dialysis was performed with different renaturation buffers and with a dialysis membrane with MWCO 1200-1400 (Orange Scientific). Dialysis were performed overnight at 4 °C except that which was performed at decreasing concentrations of urea. The used buffers had the same basic components (20 mM Tris (Sigma-Aldrich) pH 8, 0.2 M NaCl (Panreac) except one or two components:

Ø 3 L of buffer containing 0.1 % (w/v) Triton X-100 (Merck). Ø 3 L of buffer containing 0.1 % (w/v) lutensol (BASF). Ø 3 L of buffer containing 10 % (w/v) glycerol (Sigma-Aldrich). Ø 3 L of 2 buffer containing 0.1 % (w/v) lutensol (BASF) and 10

% (w/v) glycerol (Sigma-Aldrich). Ø 1 L of buffer containing 10 % (w/v) glycerol (Sigma-Aldrich)

and 4 M urea (Sigma-Aldrich); 1 L of buffer containing 10 % (w/v) glycerol (Sigma-Aldrich) and 2 M urea (Sigma-Aldrich); 1 L of buffer containing 10 % (w/v) glycerol (Sigma-Aldrich) and 2 M urea (Sigma-Aldrich); 1 L of buffer containing 10 % (w/v) glycerol (Sigma-Aldrich) and 1 M urea (Sigma-Aldrich); 1 L of buffer containing 10 % (w/v) glycerol (Sigma-Aldrich). Study of r-OmpK refolding by UV spectrophometry Renatured r-OmpK samples from different dialyses and their respective buffers were exposed to a gradual temperature increase of 4 °C to 100 °C on a dry bath, accublock Digital Dry Bath (Labnet). Absorbance values at 280 nm were measured when the temperature increase and the observed changes were analysed. Absorbance was measured on the HITACHI U-2000 spectrophotometer. Study of r-OmpK refolding by Fluorescence spectroscopy The r-OmpK samples and respective buffers were placed in a quartz cuvette and the excitation wavelengths for each of the aromatic amino acids were supplied to the Fluorescence Spectrophotometer (Varian Cary Eclipse). The fluorescence intensity was measured from 10 nm more than the wavelength of excitation to 600 nm. The temperature in the fluorescence spectrophotometer was increased and repeating the previous steps. ELISA ELISA were performed to detect the presence of antibodies possibly contained in the plasma of the fish vaccinated with DNA vaccines. For the ELISA, the protocol recommended by Aquatic Diagnostics, Lda. was followed. Different amounts of protein and different dilutions of fish plasmas were used for optimization of the assays. RESULTS AND DISCUSSION Construction of the pET21a-ompK plasmid After growing E. coli carrying the pET21a-ZZcbm64 plasmid until the early stationary phase, purification of the plasmid was performed by NZYTech Kit Gel Pure and a pET21a-ZZcbm64 solution with a concentration of 217 ng/µL was obtained. A restriction enzyme digestion reaction with Xho I and Nde I was performed to separate ZZ-cbm64 from pET21a. After the digestion reaction, the mixture was placed on an agarose gel and an electrophorese was performed (Figure 2). Since pET21a and ZZcbm64 contain different number of base pairs, purification of pET21a was achieved by agarose gel purification. The bands concerning pET21a and ZZcbm64 were well visible on the gel and with the correct number of base pairs showing that the digestion and separation of the sequences were successfully achieved. After cleaving the band of the agarose gel containing pET21a and subsequent purification, a solution of purified digested pET21a with a final concentration of 14.3 ng/µL was obtained. Hereupon, the

purification yield of pET21a was 6.6 %. To obtain ompK sequence, a PCR was performed where the DNA template used was PVAX-GFP-ompK. After conclusion of ompK amplification, a solution with final concentration of 120 ng/µL was obtained. In order to adequate ompK ends compatible with pET21a ends, so that ligation between them could occur, the ompK sequence had to be digested with Xho I and Nde I as well. Since the gel purification yield is quite low and only a few base pairs are removed at the ompK ends, the restriction enzyme digestion reaction product was purified with the NZYTech Purification of PCR products Kit. A purified digested ompK solution of final concentration of 63.5 ng/µL was achieved. Hereupon, the purification yield of ompK sequence was 52.9 %, a higher yield than the one referred above.

Figure 2 Image of the reaction product of pET21a-ZZcbm64 digestion in 1% agarose gel, electrophoresis run for 1h at 100 V. Well 1 - NZYDNA Ladder III; Well 2-pET21a-ZZcbm64 digested with Xho I and Nde I. The 5363 bp band is representative of pET21a and 630 bp is representative of the ZZ-cbm64 sequence.

Once solutions of pET21a and ompK with compatible ends were achieved, the ligation reaction was carried out whose quantities and compounds were described in the Materials and Methods (section 2.2.1). With the ligation reaction product, competent E. coli (DH5α) cells were transformed and transformed cells incubated on a plate with LB agar medium containing ampicillin so that only colonies containing pET21a-ompK could grow. For the ligation product whose reaction lasted for 4 h at room temperature (A), only one colony of transformed cells was obtained and for the ligation product whose reaction lasted overnight at 4 °C (B), 36 colonies of transformed cells were grown. The colony grown from transformation A and four colonies grown from transformation B were picked from the Petri plate and cultured individually in LB liquid medium with ampicillin. After cell growth in liquid medium, the plasmid DNA of the cells was purified. Purified plasmid DNA from each colony was digested with Eco RV. This enzyme has a cleavage site in the sequence of pET21a and in the ompK sequence. In this way, it is possible to verify the presence of pET21a-ompK if it was observed on the agarose gel two DNA bands with approximately 4839 bp and 1305 bp after digestion. As can be seen in Figure 3, the result of the Eco RV enzyme digestion of purified plasmid DNA of the cell colonies grown after transformation with the pET21a-ompK ligation was placed on an agarose gel. In lanes 2, 5 and 7 there was a visible band with approximately 1300 bp, however in well 2 there was another band with approximately 4000 bp was observed and this colony was discarded (cell from colony 1 of transformation A) as a possible candidate for pET21a-ompK transformant. As previously described, for the ligation affirmation, two DNA bands, one with approximately 1300 bp and one with approximately 5000 bp, would have to be observed. The colonies 3 and 5 derived from transformation B whose plasmid DNA digested with Eco RV was run in lanes 5 and 7 (detached with blue rectangles) were proposed as possible pET21a-ompK transformants. Subsequently, the digestion with Eco RV was repeated with greater amount of restriction enzyme to make sure that all the DNA would be well digested. In lane 8 one can observe

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the repeat of DNA digestion of colony 3 (transformation B) and in lane 9 one can observe the repeat of DNA digestion of colony 7 (transformation B). In both digested plasmid DNA, it was possible to verify the presence of a band at approximately 5000 bp and another with approximately 1300 bp. The presence of the pET21a-ompK plasmid in these two colonies (3 and 5 - transformation B) of E. coli (DH5a) was confirmed.

Figure 3 1% agarose gel with pDNA digestion of colonies transformed with the pET21a-ompK ligation with the Eco RV restriction enzyme. 1 - NZYDNA Ladder III; 2 - purified pDNA from cell colony 1 (transformation A) digested with Eco RV; 3 - purified pDNA from cell colony 1 (transformation B) digested with Eco RV; 4 - purified pDNA from cell colony 2 (transformation B) digested with Eco RV; 5 - purified pDNA from cell colony 3 (transformation B) digested with Eco RV; 6 - purified pDNA from cell colony 4 (transformation B) digested with Eco RV; 7 - purified pDNA from cell colony 5 (transformation B) digested with Eco RV; 8 - Repeated digestion of purified pDNA from cells of colony 3 (transformation B) with Eco RV; 9 - Repeated digestion of purified pDNA from cell of colony 5 (transformation B) with Eco RV. Electrophoresis was ran for 1 h at 100 V (electrophoresis power Supply-EPS 301, General Electrics).

Expression of r-OmpK and verification of its production For the expression of the ompK gene and consequently r-OmpK production, was necessary to transform another E. coli strain. Purified DNA from colony 3 (transformation B) was used to transform chemically competent E. coli BL21(DE3) cells. After transformation, the transformed cells were cultured overnight in LB solid medium supplemented with ampicillin. In the Petri dish, countless transformed colonies could be observed. Three of these colonies were picked and grown in LB liquid medium with ampicillin. Subsequently, the plasmid DNA of these grown colonies was purified with the Miniprep kit from NZYTech. Three solutions of pET21a-ompK with a final concentrations of 46.5 ng/µL, 60.0 ng/µL and 41.5 ng/µL, respectively, resulted from purification. The BL21(DE3) transformation with pET21a-ompK also had to be confirmed. The plasmid DNA from the transformed colonies was also digested with Eco RV to verify if the transformation was successful.

Figure 4 1% agarose gel with pDNA digestion of colonies of E. coli BL21(DE3) transformed with the pET21a-ompK plasmid with the Eco RV restriction enzyme.

1 – NZYDNA Ladder III; 2 - purified pET21a-ompK from cell colony 1 digested with Eco RV; 3 - purified pET21a-ompK from cell colony 2 digested with Eco RV; 4 - purified pET21a-ompK from cell colony 3 digested with Eco RV. Electrophoresis was ran for 1 h at 100 V (electrophoresis power Supply-EPS 301, General Electrics)

In Figure 4, is possible to observe the result of the Eco RV enzyme digestion of purified plasmid DNA of the E. coli BL21(DE3) cells transformed with the pET21a-ompK plasmid. In lanes 2, 3 and 4 of the agarose gel, two bands were observed, one with approximately 5000 bp and the other one with 1300 bp. Therefore, all grown colonies that were picked after transformation, confirmedly contained pET21a-ompK. The expression of r-OmpK was then followed. Cells from E. coli BL21(DE3) (negative control) and E. coli BL21(DE3)+pET21a-ompK cells were grown individually in 150 mL of LB broth. E. coli BL21(DE3), cells without pET21a-ompK, were grown deprived of the addition of ampicillin as they did not contain plasmid which would give them resistance to the antibiotic. Cell growth was followed by UV/vis spectrophotometry (wavelength 600 nm). When an optical density of 1 was achieved, IPTG was added to the broth to induce production of interest protein into cells. After induction with IPTG, the cell growth lasted for further 6 hours. Following these 6 hours the optical density was measured again and a final OD of 4 was noted. Cells were then collected by centrifugation. The supernatant was discarded. Cells present in the pellet were resuspended in 5 mL of TST buffer. The cell suspension was sonicated and then centrifuged for separation of the soluble components and the insoluble components of the cell. After centrifugation, the pellet components were solubilized in the SDS buffer. The solution of the supernatant and solution of the solubilized components of the pellet were placed on a polyacrylamide gel and run under denaturant conditions for 2h at 90 V. These procedures were performed for E. coli BL21 (DE3) and E. coli BL21 (DE3)+pET21a-ompK cells to visualize the presence of OmpK.

Figure 5 12% Polyacrylamide gels under denaturant conditions with samples of the protein extract of the supernatant and the solubilized pellet protein extract from E. coli BL21(DE3) and BL21(DE3)+pET21a-ompK sonicated cells induced with IPTG. 1 – Dual color protein ladder (Biorad); 2 – protein extract of the supernatant of BL21(DE3); 3 - protein extract of the supernatant of BL21(DE3)+pET21a-ompK; 4 - solubilized pellet protein extract from BL21(DE3); 5 - solubilized pellet protein extract from BL21(DE3)+pET21a-ompK; 6 - Page Ruler Plus Prest (Thermo Scientific); 7 - protein extract of the supernatant of BL21(DE3) diluted 1/20; 8 - protein extract of the supernatant of BL21(DE3)+pET21a-ompK diluted 1/20. Electrophoresis was run for 2 h at 90 V (Biorad).

In Figure 5, lanes 2 and 3 are representative of the extracts of the supernatants, but since the samples were heavily saturated, another gel with the same samples was run 1/20 diluted, as can be seen in the gel on the right side with lanes 7 and 8, respectively. In the well 5 (BL21(DE3)+pET21a-ompK pellet protein), it was possible to distinguish a band from the rest of the protein bands and that does not found in the BL21(DE3) pellet protein extract (lane 4). This band was lied within the range indicated by the molecular weight indicator 25-37 kDa. Since the protein of interest had 31.3 kDa

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molecular weight, it was assumed that the prominent band present less than 31.3kDa is representative of r-OmpK since the signal peptide was removed. Although the r-OmpK protein was produced and remained in the cytoplasm once the signal peptide was removed from ompK sequence, it was found in the pellet of the sonicated and centrifuged cells. It was possible to conclude that the protein is found in aggregates also known as IBs which precipitate due to their weight and size, rendering them insoluble in the plasma. Purification of r-OmpK protein The formation of protein IBs was important for its purification. The ompK sequence was introduced into pET21a plasmid with the stop codon. Although pET21a possesses a sequence capable of producing a histidine tail, since the stop codon of the ompK sequence has not been removed, the tail of histines has not been produced and is not incorporated in the OmpK protein. Three different purification methods were performed. For each method, all the supernatants from every wash and centrifugation steps were stored as well as the respective final pellets containing the IBs. For the study of the best method of purification of IBs, all stored samples were run on a polyacrylamide gel under denaturing conditions after quantification of their protein content. Before being placed on the gel (figure 6), the samples were diluted in order that they all had the same protein concentration except those having a very low protein concentration.

Figure 6 12% Polyacrylamide gels where samples of supernatants and solubilized pellets of different IBs purification methods were placed for electrophoresis under denaturing conditions. 1 - Page Ruler Plus Prest (Thermo Scientific); S – supernatant (a,b,c,..); P – pellet; C – (Negative control) protein from BL21 (DE3) sonicated cells; pET - protein from BL21(DE3) + pET21a-ompK sonicated cells. Electrophoresis was run for 3 h at 90 V (Biorad).

In the first centrifugation, some inclusion bodies (Sa-pET) were removed, although they did not seem to be a problem since in P-pET the r-OmpK protein band was highlighted. In the P-pET lane of method 1 is possible to visualize that the IBs, despite being in greater quantity, were not yet pure, requiring more washes to eliminate contaminating proteins. In method 2, in the first centrifugation also some protein of interest was transported to the supernatant. Although calculations were made for having the same amount of protein placed in all lanes, it was observed that the P-pET well of method 1 has more total protein amount than the P-pET lane of method 2. In this way, it became difficult to compare and visualize which of the IBs were purer. For method 3, washes and centrifugations did not carry r-OmpK protein out of the pellet. In the Sc-pET-method 3 lane there was a problem in the gel but it was discarded since the first centrifugations are the most relevant for the

selection of the best method. Like in the pellet of method 2, the P-pET-method 3 appeared to contain less total protein than the corresponding pellet of method 1. Furthermore, it not possible to compare visually if it contained fewer contaminants and/or a greater amount of r-OmpK protein than that of method 2. Two of the three purification methods, method 1 and method 3, were chosen because method 2 and method 3 were identical. The method 1 and method 3 for r-OmpK purification from pellets were repeated. Pellets were washed over several times. Pellets from sonicated E. coli BL21(DE3) cells without r-OmpK were discarded since they operated only to identify the protein of interest. The pellets were dissolved, total protein quantified by Pierce™ BCA Protein Assay Kit and final concentrations succeeded as 40.5 g/L and 85.6 g/L, for the pellet of Method 1 and Method 3, respectively. The pellet from Method 3 was diluted to have the same concentration as the pellet sample from Method 1, 40.5 g/L. Once having the same protein concentration, a new polyacrylamide gel under denaturing conditions was run which allowed to visualize which pellets contained more contaminants and more protein of interest. Dilutions of the dissolved pellet samples were made for the same purpose. The analysis of Figure 7 was fundamental to choose a final purification method. Since the samples from lanes 2 and 3, 4 and 5, 6 and 7, 8 and 9 were contained the same total protein concentrations, it was possible to verify that they were contained approximately the same amount of r-OmpK. Method 1 was chosen as the purification method of all the protein produced by E. coli BL21(DE3)+pET21a-ompK cells. Samples from method 3 showed a higher amount of contaminating proteins and although this method produced a greater amount of total protein, it was preferable to have a lower amount of protein with a higher purity. Although method 1 still had contaminating proteins, they were in a very small percentage in the sample.

Figure 7 12 % Polyacrylamide gel where samples solubilized pellets purified with method 1 and method 3 were placed for electrophoresis under denaturing conditions. 1- Page Ruler Plus Prest (Thermo Scientific); 2 – solubilized pellet (method 1); 3 - solubilized pellet (method 3); 4 - solubilized pellet (method 1) 1:5 dilution; 5 - solubilized pellet (method 3) 1:5 dilution; 6 - solubilized pellet (method 1) 1:10 dilution; 7 - solubilized pellet (method 3) 1:10 dilution; 8 - solubilized pellet (method 1) 1:20 dilution; 9 - solubilized pellet (method 3) 1:20 dilution. Electrophoresis was run for 3 h at 90 V (Biorad).

As r-OmpK had the final aim of performing ELISA assays, the existence of such a small amount of contaminants was not relevant since the ELISA technique is an immunoassay with high affinity, is sensitive and has high specificity. After purification of the r-OmpK IBs from all the E.coli cell culture (Vfinal = 150 mL), the protein content of the pellets was again quantified. One pellet contained 30 mg of protein and a total of 20 pellets were obtained. The total protein purified from a 150 mL growth of E. coli BL21(DE3) was then 600 mg. The dry weight of producer E. coli cells per liter of suspension was obtained by multiplying the final optical density (OD600nm = 4.0) by 0.304 and by the volume of suspension (0.304 x 4 x 0.150). Was obtained 0.182 g dry weight and consequently, 3290 mg protein/g dry weight. Analysis of r-OmpK refolding methods – UV spectroscopy For the study of protein refolding, renatured r-OmpK samples from different dialyses and their respective buffers were exposed to a

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gradual increase of temperature (4 °C to 100 °C) on a dry bath, accublock Digital Dry Bath (Labnet).

Figure 8 Representative graphic of ratios of protein absorbance values and protein initial absorbance value vs temperature rise. Blue line – r-OmpK renatured in 20 mM Tris pH 8, 0.2 M NaCl and 0.1 % (w/v) lutensol; Orange line – r-OmpK renatured in 20 mM Tris pH 8, 0.2 M NaCl and 0.1 % (w/v) Triton X-100; Grey line - r-OmpK renatured in 20 mM Tris pH 8, 0.2 M NaCl and 10 % glycerol (dialysis with progressive decrease of urea concentration); Yellow line - r-OmpK renatured in 20 mM Tris pH 8, 0.2 M NaCl, 0.1 % (w/v) l utensol and 10 % glycerol; Green line - r-OmpK renatured in 20 mM Tris pH 8, 0.2 M NaCl and 10 % glycerol.

Absorbance values at 280 nm were measured and the observed changes were analysed. Absorbances of both buffer containing r-OmpK and buffer without r-OmpK samples were measured. The value obtained for the respective buffer was subtracted from the values obtained from the readings of the samples containing protein. Therefore, it was possible to eliminate interferences that could be present in the buffers. To have a better understanding of how much the absorbance values increased since the protein was subjected to a temperature rise and consequently exposing its aromatic amino acids, absorbance ratios were performed. The corrected absorbance value corresponding to each temperature was divided by the initial corrected absorbance value. Observing figure 8, as expected, in most assays where the temperature was increased there was also an increase in absorbance ratios except for r-OmpK renatured in 20 mM Tris pH 8, 0.2 M NaCl and 0.1% (w / v) Triton X -100. As noted above, the increase of light absorption is explained by the greater exposure of the aromatic amino acids with denaturation of the protein. Aromatic amino acids such as phenylalanine, tyrosine, and tryptophan have hydrophobic chains that tend to confine within proteins. These amino acids are shielded by other more hydrophilic amino acids. Consequently, they were protected from UV light not absorbing it. When exposed, UV light can already reach the aromatic chains of these amino acids, thereby increasing the absorbance value registered by the equipment. Since the studied protein was the same and was in the same concentration for all the five cases, the number of amino acids with UV absorption properties in the range of 280 nm were identical. It was speculated that the greater the difference between the absorbance ratio at 4 °C and the absorbance ratio at 100 °C, more tertiary structure would be the r-OmpK after its denaturation. Thus, analysing the figure 8, the best method for protein refolding was the dialysis with buffer containing r-OmpK renatured in 20 mM Tris pH 8, 0.2 M NaCl and 0.1 % (w/v) lutensol. Analysis of r-OmpK refolding methods – Fluorescence spectroscopy The excitation wavelength of the tryptophan was 280 nm and the expected emission wavelength was 348 nm, the excitation wavelength of the tyrosine was 274 nm and the expected emission wavelength was 303 nm and the excitation wavelength of the phenylalanine was 257 nm and the expected emission wavelength was 282 nm. For an excitation wavelength of 280 nm, both Trp and Tyr were exited since their excitation wavelengths are very similar. To excite Trp exclusively, it should have been used a wavelength of 295 nm. However, since the two amino acids were excited, it was observed a

higher signal since the r-OmpK has only 6 tryptophan but 18 tyrosine. Fluorescence assays were performed with the same concept as that performed on UV spectrophotometry. In this sense, it was expected that with the increase of temperature there would also be an increase in the emission of fluorescence by hydrophobic amino acids exposure. Assays at 257 nm (phenylalanines excitation wavelength) were also carried out in order to study the refolding of the r-OmpK by the FRET technique. Föster resonance energy transfer (FRET) is a process in which a donor molecule in excited state transfers its excitation energy through dipole-dipole coupling to an acceptor fluorophore, when the two are brought into proximity (typically less than 10 nm) (Saraheimo et al., 2013). Upon excitation at a characteristic wavelength the energy absorbed by the donor (in this case an amino acid) is transferred to the acceptor (another amino acid), which in turn emits the energy (Saraheimo et al., 2013). If the amino acids were close, there was a tertiary structure of r-OmpK, when the excitation wavelength was 257 nm the emitted fluorescence should be shifted to 348 or 303 nm instead of 282 for phenylalanines. With the increase of temperature and consequently denaturation of the protein that should separate the amino acids and the emitted fluorescence was expected with a wavelength of 282 nm. In the figure 9, it is possible to observe the fluorescence emission graphics of refolded r-OmpK dialyzed with 20 mM Tris pH 8, 0.2 M NaCl, 0.1 % (w/v) lutensol and exposed to different temperature. When the protein was excited at 280 nm (A), a peak at approximately 337 nm for each temperature can be detected. As mentioned above, 303 and 348 nm are the emission wavelength of Tyr and Trp respectively. As at 280 nm we excited Tyr and Trp, the wavelength at which the maximum fluorescence intensity was detected (337 nm) can correspond to the overlap of the fluorescence emitted by the two amino acids. With the increase of temperature and consequently denaturation of the r-OmpK, the fluorescence intensity was expected to increase once the amino acids with aromatic chain which had capable of emitting fluorescence were being exposed. However, it was possible to observe that exactly the opposite occurred, the fluorescence intensity decreased when temperature rise. After several research, it was possible to perceive this occurrence is quite common since this technique of spectroscopy has environmental factors that can affect in the fluorescence emission such as interactions between the fluorophore and surrounding solvent molecules (solvent polarity), dissolved inorganic and organic compounds, temperature, pH, and other. Fluorescence quantum yield is the fraction of the number of quanta absorbed by a molecule that are emitted as fluorescence. The parameters described above can heavily influence the quantum yield of a molecule. In the study of proteins, where aromatic amino acids were excited, the polarity of the surrounding environment may be the most relevant factor for the decrease of quantum yield and consequently the decrease of fluorescence intensity. In solution, polar solvent molecules surrounding the ground state fluorophore have dipole moments that can interact with the dipole moment of the fluorophore. Energy level differences between the ground and excited states in the fluorophore produce a change in the molecular dipole moment, which ultimately induces a rearrangement of surrounding solvent molecules. Solvent relaxation is when solvent molecules assist in stabilizing and further lowering the energy level of the excited state by reorienting around the excited fluorophore. This has the effect of reducing the energy separation between the ground and excited states. Increasing the solvent polarity produces a correspondingly larger reduction in the energy level of the excited state, while decreasing the solvent polarity reduces the solvent effect on the excited state energy level. In proteins where the fluorophores are the aromatic amino acids, when it has a tertiary structure, the fluorophores are usually located inside the protein where the

8

Figure 9 Figure 10 Fluorescence emission graphics of refolded r-OmpK with dialysis buffer containing 20 mM Tris pH 8, 0.2 M NaCl, 0.1 % (w/v) lutensol. A – Fluorescence emission of protein when it was excited with UV light at 280 nm; B - Fluorescence emission of protein when it was excited with UV light at 257 nm (FRET). In both graphics, different colours of lines represent a fluorescence emission of the molecules of protein when it was exposed to a specific temperature. Dark blue line – protein at 4 °C; Light blue line – protein at 20 °C; Yellow line – protein at 40 °C; Orange line – protein at 60 °C; Red line – protein at 80 °C; Dark red line – protein at 100 °C

environment that surrounds them is hydrophobic and where the polarity is usually lower and the quantum yield is higher. When protein is denatured, in this case with the increase of temperature, the aromatic chains of the amino acids are exposed to the aqueous solvent which is polar. The polar environment causes a decrease of quantum yield thereby decreasing the intensity of fluorescence. (Hawe et al.,2008) If the reason for a decrease in fluorescence intensity is that mentioned above, it is possible to affirm that before denaturation of the protein, r-OmpK had a tertiary structure once it emitted more fluorescence than after denaturation. It was also possible to observe in the Figure 9 that as the protein was denatured, the peak of fluorescence intensity was shifted slightly to lower wavelengths. Since r-OmpK had higher Tyr content than Trp, when all amino acids were exposed, the fluorescence intensity of the excited Tyr was higher. In this way, the fluorescence intensity peak was shifted to the emission wavelength of this amino acid. When the protein was excited at 257 nm (B), a peak at approximately 348 nm for each temperature can be detected too. Since the wavelength of the observed peak was corresponding to the Trp emission wavelength, it was possible to affirm that the FRET process occurred, where the emission energy of the excited phenylalanines was able to excite the tryptophanes. If this process occurred, the amino acids were close enough, showing some tertiary structure. As in Chart A, there was a decrease in fluorescence intensity level when the temperature was increased. A peak fluorescence intensity shift was predicted from 348 nm to 282 nm as the temperature rise and consequently the amino acids separation with denaturation of the protein. With the study of the protein when refolded in the other buffer (dialysis buffer containing 10 % glycerol, dialysis buffer containing 0.1% lutensol and 10 % of glycerol; dialysis buffer containing 10% glycerol (decrease concentration of urea) was understood that r-OmpK behaved very similarly although refolded in different dialysis buffers. Protein refolded in buffer containing 0.1 % Triton X-100 was shown to have no structure. The greatest difference was observed in the values of the fluorescence intensities where the order of the fluorescence graphics where the values of the peaks of intensity go from the highest to the lowest corresponds to the values verified by UV spectrophotometry too. r-OmpK renatured in buffer with lutensol had a peak of greater fluorescence intensity, then renatured in buffer containing glycerol + lutensol, renatured in buffer with glycerol (dialysis with progressive decrease of urea concentration), renatured in buffer with glycerol and finally renatured in dialysis buffer with triton X-100 where almost no structure has been verified. Since the protein refolded in buffer containing 20 mM Tris pH 8, 0.2 M NaCl, 0.1 % (w/v) Lutensol showed to have higher absorption in UV spectroscopy and higher intensity of fluorescence emission in fluorescence spectroscopy and thus more tertiary structure, the r-OmpK achieved under these

conditions was used for the ELISA. One limitation of the tests used to study the protein is whether the structure achieved was the native structure of OmpK. Denatured protein may be present, but the hydrophobic moieties are "closed" in pockets and with the increase of temperature the solubility is increased too and can lead to the opening of these pockets. To verify if the achieved structure was the native, other techniques for studying the protein had to be performed, for example, nuclear magnetic resonance (NMR). Once the ELISA assays are very sensitive, the samples of r-OmpK renatured in buffer containing lutensol was then quantified to know the exactly concentration of the protein. The concentration of the protein sample obtained by Pierce™ BCA Protein Assay Kit was 100 µL/mL. The volume of the analysed sample was 10 mL per pellet. There were 20 pellets of proteins that resulted in a final volume of 200 mL protein solution and 20 mg of total protein. The dry weight of grown E. coli was obtained by multiplying the final optical density (600 nm) to 0.304 and to volume of growth (0.306 x 4 x 0.150). Was obtained 0.1824 g dry weight and consequently, 110 mg protein/g dry

weight. Before the performance of the dialyses, it was obtained 3290 mg protein/g dry weight showing a yield of only 3.34 % of renatured protein. During dialysis, much of the protein may be precipitated, and this may be one of the reasons for achieving such a low final protein concentration. ELISA In this experimental work, the objective of ELISA was to detect the presence of antibodies possibly contained in the serum of the fish vaccinated with DNA vaccines. ELISAs were performed as described in the section 2.9 such as the use of fish plasmas and r-OmpK protein amounts in the 96-wells plate. Plasma from three fish vaccinated with pVAX-GFP (positive control) and plasma from three fish vaccinated with pVAX-GFP-ompK were tested firstly. Analysing the Figure 11-A, as expected, when only 0.2 µg of protein was adhered to the plate the absorbance values were lower except when was used 1/40 diluted fish sera. When 1.0 and 2.0 µg of r-ompK were used, the absorbance values were increased. Comparing the results, for solid dark blue bars and dashed dark blue bars, regardless of the amount of protein used, the absorbance values should be equal since no fish plasmas were used and the Aquatic diagnostics antibody should not have reacted with r-OmpK. For solid light blue bars and dashed light blue bars (1/5 diluted fish plasmas), when the amount of protein was increased, the absorbance values were increased too however, when 0.2 and 1.0 µg of protein were used, the values of absorbance for fish vaccinated with pVAX-GFP-ompK plasmas were lower than for fish vaccinated with pVAX-GFP. For solid yellow bars and dashed yellow bars (1/40 diluted fish plasmas), when the amount of protein was increased, the absorbance values were first decreased and then increased and a

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Figure 11 A- Absorbances (445 nm) measured from the 96 wells plate of the ELISA. Two assays were performed, one for fish vaccinated with pVAX-GFP-ompK plasmas and one for fish vaccinated with pVAX-GFP plasmas. For each assay, plasma from three different fish and three different amounts of r-OmpK protein (0.2, 1.0 and 2.0 µg) were used. Solid dark blue bar - assay without plasmas of fish vaccinated with pVAX-GFP-ompK; Dashed dark blue bar - without plasmas of fish vaccinated with pVAX-GFP; Solid Light blue bar- assays performed with 1/5 dilution of fish plasmas vaccinated with pVAX-GFP-ompK; Dashed Light blue bar - assays performed with 1/5 dilution of fish plasmas vaccinated with pVAX-GFP; Solid Yellow bar - assays performed with 1/40 dilution of fish plasmas vaccinated with pVAX-GFP-ompK; Dashed Yellow bar - assays performed with 1/40 dilution of fish plasmas vaccinated with pVAX-GFP; Solid red bar- assays performed with 1/320 dilution of fish plasmas vaccinated with pVAX-GFP-ompK; Dashed red bar - assays performed with 1/320 dilution of fish plasmas vaccinated with pVAX-GFP. B - Ratio between the absorbances (445 nm) measured from the 96 wells plate of the ELISA of fish vaccinated with pVAX-GFP-ompK plasmas and ELISA of fish vaccinated with pVAX-GFP plasmas. Dark blue bar - assays performed without plasmas of fish; Light blue bar – assays performed with 1/5 diluted fish plasmas; Yellow bar - assays performed with 1/40 diluted fish plasmas; Red bar - assays performed with 1/320 diluted fish plasmas.

correlation of the values was not achieved. When 2.0 µg of protein was used, the values of absorbance for fish vaccinated with pVAX-GFP-ompK plasmas were lower than for fish vaccinated with pVAX-GFP. For solid red bars and dashed red bars (1/320 diluted fish plasmas), when the amount of protein was increased, the absorbance values were first increased and then decreased and a correlation of the values was not achieved too. Regardless of the amount of protein used, the values of absorbance for fish vaccinated with pVAX-GFP-ompK plasmas were always higher than for fish vaccinated with pVAX-GFP. It was expected that the absorbances would increase as less plasma dilution was used but this assertion was not observed. The obtained error values showed large variability of the absorbance values and made difficult to take accurate conclusions.

Figure 12 Absorbance average of ELISA performed with 1.0 µg of r-OmpK and 1/40 diluted fish plasmas. Dark blue bar - assays performed using plasmas from 16 fish vaccinated with pVAX-GFP-ompK; Light blue bar – assays performed using plasmas from 16 fish vaccinated with pVAX-GFP; Yellow bar - assays performed using plasmas from 8 fish vaccinated with pVAX-GFP-frag.ompK; Red bar - assays performed using plasmas from 8 fish vaccinated with PBS.

In order to make easier to compare results, the ratios between the absorbances obtained from the evaluation of the plasmas of the fish vaccinated with pVAX-GFP-ompK and the absorbances obtained from the evaluation of plasmas from the fish vaccinated with pVAX-GFP were calculated (Figure 11-B). When values of the bars were above 1.0 the absorbances from assays using fish vaccinated with pVAX-GFP-ompK plasmas were higher than absorbances from assays using fish vaccinated with pVAX-GFP plasmas. When this happened, it meant that the vaccinated fish had larger amounts of antibodies that react with r-OmpK.

Observing the Figure 11-B, it was possible to see that the absorbance of assays using fish vaccinated with pVAX-GFP-ompK plasmas was much greater than the absorbance of assays using fish vaccinated with pVAX-GFP plasmas were those performed with 1 µg of r-OmpK and 1/40 dilution of the plasmas. This condition was then used to perform another ELISA using plasmas of a higher number of vaccinated fish. It was tested plasmas from 16 fish vaccinated with pVAX-GFP-ompK, plasmas from 16 fish vaccinated with pVAX-GFP, plasmas from 8 fish vaccinated with pVAX-GFP-frag.ompK and plasmas from 8 fish vaccinated with PBS were tested (Figure 12).

Figure 12 Ratio between the absorbances (445 nm) measured from the 96 wells plate of the ELISA of fish vaccinated with pVAX-GFP-ompK, pVAX-GFP-frag.ompK and PBS plasmas and ELISA of fish vaccinated with pVAX-GFP plasmas. Dark blue bar – absorbance of assays performed with plasmas from fish (pVAX-GFP-ompK)/ absorbance of assays performed with plasmas from fish (pVAX-GFP); Light blue bar – absorbance of assays performed with plasmas from fish (pVAX-GFP-frag.ompK)/ absorbance of assays performed with plasmas from fish (pVAX-GFP); Red bar - absorbance of assays performed with plasmas from fish (PBS)/ absorbance of assays performed with plasmas from fish (pVAX-GFP).

Observing the Figure 12, it was possible to notice that the measured absorbance values were higher for the assays where was used plasmas from fish vaccinated with pVAX-GFP. This plasmid does not have the ompK gene sequence showing that it was not observed immunological difference between fish vaccinated with plasmid containing the ompK sequence, fish vaccinated with plasmid containing the part of ompK sequence and fish vaccinated with pVAX-GFP or PBS.

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In order to make easier to compare results, the ratios between the absorbances obtained from the evaluation of the plasmas of the fish vaccinated with pVAX-GFP-ompK, pVAX-GFP-frag.ompK and PBS and the absorbances obtained from the evaluation of plasmas from the fish vaccinated with pVAX-GFP were calculated (Figure 13). When values of the absorbances from assays using fish plasmas were lower than 1.0 means that the sera samples had contained less amount of antibodies that react with r-OmpK that the sera samples from fish vaccinated with pVAX-GFP concluding that the vaccine was not efficient. However, as in previous assays, the obtained error values showed large variability of the absorbance values and made difficult to take accurate conclusions. Plasmas collected after 4 weeks from the vaccination day were only analysed. The plasmas collected after 8 weeks from the day of vaccination were not analysed. Although the term ‘humoral memory’ has been employed to refer to the persistent antibody response (even that elicited during the primary response it is important an exposure of a secondary antigen to elicit a differentiated response to that antigen (Ye et al., 2013). Since a vaccination boost was not administrated in fish, the analysis latter plasmas results are expected to be the same as for the plasmas collected after 4 weeks from the day of vaccination. Fish vaccination with naked DNA may be one of the reasons why the plasmid did not function as a vaccine since the DNA may not have entered the cells. The study of new methods of plasmid delivery to cells such as the use of biocompatible nanoparticles would be very important for the development of a final and efficient vaccine. Conclusions Vaccination with outer membrane protein such as OmpK (one of major OMPs and widely distributed among Vibrio) has been shown to be effective against some Vibrio spp infections in fish due to its capacity to initiate an immune response in host cells. The conserved OmpK of V. alginolyticus ZJ04107 is an effective vaccine candidate against infection by V. alginolyticus. Fish immunized intraperitoneally with the OmpK produced high levels of anti-OmpK antibodies. (Qian et al., 2008). However, antigens which are purified and structurally matured are important for effective immunization. Protein purification with its structural mature form involves many processes and it is difficult to obtain. DNA vaccines offer several advantages over classical antigen vaccines including an easily purification. DNA vaccines using omp genes have already been shown to give partial protection against V. anguillarium (Kumar et al., 2007). V. alginolyticus is one of the main pathogenic Vibrios to marine animals, and leads to large economic damage in marine aquaculture and to develop an effective vaccine against it is necessary. The research done by Kumar may serve as an aid to the development of the DNA vaccine against Vibrios infection using ompK gene. The gene encoding to OmpK protein from Vibrio was cloned in a pVAX-GFP vector, thereby transformed E. coli cells had been selected and first plasmid vaccine candidates had been produced and purified at a scale proper for in vitro transfection studies and in vivo fish immunization trials. This experimental work had been done before the current work started. Solea senegalensis is a common high-value flatfish in Southern Europe, commonly reared in extensive aquacultural production in Portugal and Spain (Dinis et al., 1999). Juveniles were selected for vaccination trials with the DNA vaccine and control solutions. The vaccination and plasmas recovery were executed in the investigation center CETEMARES located in Peniche by Professor Teresa Baptista. The results of immunization levels were evaluated. To test the efficiency of the vaccine it was necessary to perform several laboratory experiments including the production, purification and refolding of the antigen, OmpK protein.

First, the ompK gene was introduced in pET21a plasmid. The plasmid had contained another gene, ZZ-cbm64, that had to be removed. After the digestion reaction with the restriction enzymes (Xho I and Nde I) to discard ZZ-cbm64 gene, pET21a was purified and a ligation reaction with ompK gene was followed. E. coli (DH5a) competent cells were transformed with the ligation product and cultured in a Petri plate. The grown colonies were picked and their plasmid DNA was extracted and digested with Eco RV restriction enzyme. The digestion reaction was done to prove the transformed cells contained the pET21a-ompK. Two of six picked colonies showed that the desire transformation with the plasmid with ompK gene was achieved. Therefore, bacteria containing pET21a-ompK were grown and E. coli BL21(DE3) competent cells were transformed and cultured in a Petri plate. Three of three picked colonies revealed to contain pET21a-ompK plasmid when plasmid was purified and digested with Eco RV. These bacteria were cultured and the protein production was induced by adding IPTG. By sonication, the cells were then disrupted and, as expected, the r-ompK formed inclusion bodies. This fact was important to the purification steps. Out of three methods executed for the protein purification, the method 1 - 1 % SDS solution, DNAse I and MgSO4 followed by successive washes with 50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl and 0.5 % (w/v) Triton X-100 and centrifugation steps - showed to be the best method since a higher amount of purer protein was collected. After purification, the total protein was quantified. The total protein purified from a 150 mL culture of E. coli BL21(DE3) was 600 mg that resulted in 3290 mg protein/g dry weight cells. The protein was found in IBs and had to be denatured and subsequently renatured. Protein renaturation was a challenge since r-OmpK did not have histidine tail and most of the protocols found in the literature for protein refolding were implemented on nickel columns. The principle of the refolding techniques was the removal of the denaturing agent from the solution where the protein was dissolved, the protocol was adapted and the denaturation agent was removed by dialysis. Different dialysis buffers were used to attempt OmpK return to its native tertiary structure. UV spectrophotometry and fluorescence spectroscopy studies allow to assume that r-OmpK renatured in 0.1% lutensol buffer presented higher level of folding. When protein was subjected to temperature rise, the absorbance values were increased and fluorescence emission intensity were decreased due to exposure of the aromatic amino acids. It was expected that fluorescence emission intensity would have increased as happened with absorbance values, but since aromatic amino acids are often found in hydrophobic and apolar environments inside proteins, when exposed to the aqueous solvent as a result of denaturation, the quantum fluorescence yield of amino acids decreased with interaction of the polar solvent surrounding them. The difference between the refolded protein absorbance and fluorescence emission intensity for the protein when fully denatured was more accentuated when renatured in buffer containing 0.1 % lutensol and this led to the assumption that r-OmpK presented higher level of folding in presence of this surfactant. Although it has been assumed that the protein has gained a complete or partial tertiary structure. However, to fully verify that fact, structural analysis techniques as NMR or X-ray crystallography would be needed. Denatured protein may be present, but the hydrophobic moieties can be "closed" in pockets and with the increase of temperature the solubility is increased too and can lead to the opening of these pockets. The total protein purified and refolded from a 150 mL culture of E. coli BL21(DE3) was 20 mg that resulted in 110 mg protein/g dry weight of cells showing a yield of only 3.34 % of renatured protein. The yield of the refolding process was quite low and was supposed that the protein precipitated and a small percentage of protein was soluble in the

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buffer. In this way, the buffer used had to be improved in order to decrease the tendency of OmpK to precipitate. Lastly, the protein refolded in lutensol buffer was used to perform immunological tests as antigen of antibodies possibly produced by vaccinated S. senegalensis. The ELISAs were executed in a very preliminary way to optimize the protocol for future assays. Considering these preliminary assays, the use of 1/40 diluted fish plasmas and 1 µg r-OmpK per well were concluded to be the best parameters to perform the assays. Subsequently, assays were performed with these conditions using plasma from 16 fish vaccinated with pVAX-GFP-ompK, plasma from 16 fish vaccinated with pVAX-GFP, plasma from 8 fish vaccinated with pVAX-GFP-frag.ompK and plasma from 8 vaccinated fish with PBS and the results demonstrated that naked ompK gene vaccination did not show increased immunity of the vaccinated fish at 4 weeks post-vaccination. References Actis, L. A., Tolmasky, M. E., & Crosa, J. H. (1999). Fish diseases

and disorders, viral, bacterial and fungal Infections. In Vibriosis (eds Stevenson, RM & Woo, PT), 523-557.

Alpar, H. O., Papanicolaou, I., & Bramwell, V. W. (2005). Strategies for DNA vaccine delivery. Expert Opinion on Drug Delivery, 2(5), 829-842.

Austin, B., & Zhang, X. H. (2006). Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates. Letters in applied microbiology, 43(2), 119-124.

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