Brucellosis is an important zoonotic disease endemic in many parts of the world. Its control and prevention require safe and effective vaccines. Currently available vaccines have certain inherent drawbacks necessitating the need to improve or develop new vaccines. In this study, the sodC gene of Brucella abortus 544, which encodes for Cu-Zn SOD protein, was amplified by polymerase chain reaction (PCR) and cloned in a prokaryotic system. The recombinant protein was induced and purified by nickel affinity chromatography under denaturing conditions, refolded, and used to raise hyperimmune sera in rabbits. Western blot analysis demonstrated a specific reaction between the purified recombinant Cu-Zn SOD and hyperimmune serum, confirming the antigenicity. These results suggested the potential of Cu-Zn SOD as a vaccine candidate.

Brucella abortus, sodC gene, Cu-Zn SOD, Cloning, Expression.

Brucellosis is one of the most common bacterial zoonotic diseases caused by the Gram-negative, facultative intracellular organism, Brucella. It poses significant public health and economic concerns globally, particularly in livestock-intensive regions (Corbel, 2006). In livestock, brucellosis causes chronic infection, abortion, orchitis, reproductive failure, and reduced productivity. Humans are not the natural host of Brucella, and they acquire infection from infected animals through direct contact with infected animals or their products. There is no licensed vaccine available for human use against brucellosis, and, therefore, control and prevention can only be attained by controlling the disease in animals either through test and slaughter or vaccination (Lalsiamthara and Lee 2017).

Currently, used live attenuated vaccines, such as B. abortus S19, B. abortus RB51, and B. melitensis Rev 1, have several limitations, such as interference with serological tests, reduced efficacy, and safety concerns (Moreno, 2014; Lalsiamthara and Lee 2017; Wang et al., 2023). These limitations necessitate the development of alternative vaccine strategies, including subunit vaccines, which offer improved safety and efficacy (Yang et al., 2013; Wang et al., 2023). Among various antigens, Cu-Zn SOD, encoded by the sodC gene, has been identified as immunogenic, capable of eliciting a protective immune response (Tabatabai and Hennager 1994; Al-Dahouk et al., 2006). Previous studies have explored its potential as a vaccine candidate in different delivery systems, including DNA/RNA vaccines (Munoz-Montesino et al., 2004; Onate et al., 2003; Onate et al., 2005; Saez et al., 2008; Singha et al., 2008), live vectored vaccines (Onate et al., 1999; He et al., 2002; Saez et al., 2012), and as liposome-encapsulated vaccines (Singha et al., 2008). Kim et al. (2019) employed a Salmonella-based delivery system expressing Omp3b, BCSP31, and SOD proteins of Brucella in goats. Here, the IgG and interferon gamma concentration were significantly raised and provided protective effects provides against intraconjunctival challenge with B. abortus 544 virulent strain. Recently, Wang et al. (2023) employed a multiepitope subunit vaccine consisting of six cytotoxic T cell (CTL) epitopes, seven T helper cell (HTL) epitopes, and four linear B cell epitopes from CU/ZN-SOD, Omp31, and BP26 genes of Brucella.  The subunit vaccine was evaluated using various bioinformatics tools and by mice experimentation. This multiepitope subunit vaccine was found to induce host immune responses and confer specific protective effect in challenge studies. 

In this study we aimed to clone, express, and purify the sodC gene of B. abortus 544 and evaluate its antigenic potential for use as a recombinant protein-based subunit vaccine.

Bacterial strains. The Brucella abortus 544 isolate available in the Brucella Laboratory, Division of Veterinary Public Health, ICAR - Indian Veterinary Research Institute, was used in the study. It was grown on trypticase soy agar for 48 h at 37°C in the presence of 5% carbon dioxide. E. coli DH5a cells were used as the host strain for cloning and grown in Luria Bertani (LB) broth at 37°C with or without ampicillin (100 µg/ml).

Cloning and expression of sodC gene. The genomic DNA was extracted from B. abortus 544 using the DNAeasy blood and tissue kit (Qiagen) following the manufacturer’s protocol. The quality of the DNA was analyzed by 0.8% agarose gel electrophoresis and stored at -20°C until use. Primers specific for the sodC gene of Brucella were designed with EcoRI and HindIII restriction sites based on available sequences in GenBank; F: 

5’-AGTAGAATTCATGAAGTCCTTATTTATTGCATCG-3’ and R: 5’-CGCTAAGCTTTTATTCGATCACGCCGCAG-3’. 

The PCR amplification of the sodC gene was carried out in a 25 μl reaction volume containing 10X DreamTaq buffer, dNTP mixture (2 mM each), primers (10 pmol/μl), DreamTaq DNA polymerase (5 U/μl), and genomic DNA. Amplification conditions were as follows: an initial denaturation of 94°C for 5 min followed by 34 cycles of denaturation at 94°C for 60 s, annealing at 61°C for 45 s, and extension at 72°C for 40 s, with a final extension at 72°C for 10 min. Following amplification, the products were analyzed by electrophoresis in agarose gel (1.5%) and documented. The PCR-amplified sodC gene was electrophoresed in 0.8% agarose gel and purified using a gel extraction kit (Qiagen). The prokaryotic expression vector pPROExHTa and purified insert (sodC) were subjected to double restriction enzyme (RE) digestion with EcoRI and HindIII (Fermentas) and purified by gel extraction. The digested vector and insert were ligated using T4 DNA ligase (Fermentas, Lithuania) at 22 °C for 1 h in a thermal cycler (Eppendorf, Germany). The ligation product was transformed into E. coli DH5α competent cells prepared by the calcium chloride method (Sambrook and Russell 2001) and plated onto LB agar plates containing 100 µg/ml ampicillin. Colonies appearing on the agar plates were screened for the presence of sodC gene-specific insert by plasmid isolation, resection enzyme analysis, and sequencing.

The expression conditions were standardized by inducing the clones with varying concentrations of IPTG (0.5-1.5 mM), and the induced cells were collected at hourly intervals and analyzed by 15% SDS-PAGE to determine the optimum conditions for the expression of Cu-Zn SOD recombinant protein. Bulk induction was carried out in 1 liter of culture volume with optimized conditions. Cells were pelleted by centrifugation and stored at -20°C until used. The recombinant protein with a His-tag at its N terminal was purified by nickel affinity chromatography under denaturing conditions using a Ni-NTA column (Qiagen, Germany). Briefly, the induced cell pellet was thawed on ice for 15 min, re-suspended in 4 volumes of lysis buffer containing urea (NaH2PO4 20mM, NaCl 1 M, and urea 8 M) by repeated pipetting, and incubated on ice for 30 min. PMSF (1 mM) was added, followed by sonication (Soniprep 150) for 6 bursts of 10 s at 15 mA with a 10 s cooling period between bursts. The lysate was centrifuged at 24,100 g for 15 min at 4°C, and the supernatant was collected, loaded on a nickel agarose column, and allowed to bind. The flow-through was also collected and stored at -20°C for analysis. The column was washed twice with wash buffer (8M urea, 20mM NaH2PO4, 0.1M Tris-Cl; pH 7.4), and the bound protein was eluted with stepwise increasing gradients of imidazole (10, 20, 40, 80, 150, and 200 mM) prepared in wash buffer. The flow-through and different fractions of eluted protein were analyzed by SDS-PAGE (15%). The eluted Cu-Zn SOD recombinant protein was dialyzed (10 kDa membrane) at 4°C against decreasing concentrations of urea, viz., 6M, 4M, 2M, 1M, and 0.5M prepared in phosphate buffer saline (PBS) and finally against plain PBS to enable gradual refolding of the protein. The concentration of the purified protein was estimated by Lowry’s method (GeNeI India) against a standard curve of BSA and stored at -80°C until used.

Antigenic potential analysis. The hyperimmune serum against the recombinant Cu-Zn SOD protein was raised in three apparently healthy adult New Zealand White rabbits procured from Laboratory Animal Resources (LAR), IVRI. The rCu-Zn SOD protein (100 µg) was emulsified with Freund’s incomplete adjuvant (IFA) and injected subcutaneously at multiple sites. A booster dose of the antigen with IFA was given on day 14, and the blood was collected 4 weeks post-immunization. The serum was separated and stored at -20°C. Production of antigen-specific antibody was analyzed by western blot analysis (Towbin et al., 1979). Briefly, the purified rCu-Zn SOD protein was subjected to SDS-PAGE along with a pre-stained protein molecular weight marker and electroblotted onto a nitrocellulose membrane (NCP) using transfer buffer (Tris 0.1M, Glycine 0.192M, and 5% methanol) by the tank transfer method (GeNeI, India) at 90 volts for 1 h. The unbound site on NCP was blocked with 5% skim milk powder in Tris buffered saline with Tween 20 (TBST; Tris 50 mM, NaCl 150 mM, and 0.2% Tween 20) for 2 h at 37°C. Membranes were incubated at 37°C for 1 h with a 1:500 dilution of hyperimmune serum raised against recombinant proteins in rabbits. After incubation with primary antibody, the blot was washed thrice with TBST and incubated with a 1:2000 dilution of HRP-conjugated goat anti-rabbit IgG for 1 h at 37°C. The blots were finally washed thrice with TBST and developed with diamino-benzidine (Sigma, USA) (6 mg/10 ml of 50 mM Tris buffer; pH 7.6) in the presence of 4 µl of H2O2 (30%).

Brucellosis is endemic in many countries, causing diseases in both animals and humans (Gupta et al., 2012). Control of the disease required effective and safe vaccines; the currently available vaccines have certain shortcomings. The S19 vaccine is used only in calves, and it is not used for vaccinating adult cows, as it could result in orchitis in males, prolonged infection, and possible abortion complications in pregnant female cattle, while the RB51 vaccine has a low protective efficacy (Lalsiamthara and Lee 2017; Deng et al., 2019). In addition, these vaccines also interfere with the serological tests that are used in screening the animals.

Several different approaches have been adopted for the development of effective and safe vaccines against brucellosis. Subunit vaccines are one such approach that are considered an alternative to live attenuated vaccines, but in the development of subunit vaccines, identification of immunogenic antigens capable of eliciting protective immune responses is important. Other approaches include knockout mutants, DNA vaccines, mucosal vaccines, and live vectored vaccines, with varying degrees of success (Lalsiamthara and Lee 2017).

Brucella is an intracellular pathogen capable of replicating in both professional phagocytes and non-phagocytic cells. This ability to resist reactive oxygen intermediates generated by host cells (macrophages) is facilitated by the production of cytoplasmic Mn SOD (Sriranganathan et al., 1991) and periplasmic Cu-Zn SOD (Beck et al., 1990). The Cu-Zn SOD plays a role in virulence and immune evasion in Brucella. Research has demonstrated that the Cu-Zn SOD encoded by the sodC gene is an immunodominant antigen capable of eliciting robust T-cell responses. Tabatabai and Hennager (1994) had reported antibodies against Cu-Zn SOD in cattle serologically positive for Brucella. Bricker et al. (1990) demonstrated that, of all the classical Brucella species and biovars examined, only B. neotomae and B. suis biovar 2 did not express detectable levels of SOD. Moustafa et al. (2010) reported that B. neotomae expressed SOD at a substantially reduced level because of the presence of a single-nucleotide insertion in the promoter region of the sodC gene.

The Cu-Zn SOD protein of Brucella has been found to be a major T-cell antigen capable of inducing a protective response against Brucella (Singha, 2006). Al-Dahouk et al. (2006) identified Cu-Zn SOD as an immunogenic protein by immuno-proteomic analysis of Brucella abortus 1119-3. Thus, it is one of the promising targets for subunit vaccine development. Recent advancements in vaccine delivery methods, such as oral immunization with recombinant Lactococcus lactis expressing Cu-Zn SOD (Saez et al., 2012) and DNA vaccines co-expressing Cu-Zn SOD and cytokines like IL-2 (Gonzalez-Smith et al., 2006), have shown promising results.

In the present study, genomic DNA extracted from B. abortus 544 was used as a template for amplification of the sodC gene using specifically designed primers. Amplification of the gene produced a specific amplicon of around 522 bp (Fig. 1). The PCR-amplified sodC gene fragment and the plasmid of the prokaryotic expression vector pPROExHTa were subjected to RE digestion with EcoRI and HindIII restriction enzymes, ligated, and transformed into E. coli DH5α competent cells and plated on LB agar plates containing ampicillin. Colonies appearing on the LB agar plate after overnight incubation at 37°C were picked up randomly, inoculated into 5 ml of LB broth containing ampicillin, and propagated overnight at 37°C.

Fig. 1. PCR amplification of sodC gene. The expected amplicon size of 522 bp is observed following electrophoresis on a 1.5% agarose gel.

Plasmid isolated from rCu-Zn SOD clones was subjected to double RE digestion with EcoRI and HindIII, and the release of a specific-sized insert of around 522 bp was observed (Fig. 2). Sequencing the positive clone with the vector-specific universal primer (M13-pUC rev -49) also showed the presence of a specific insert in the correct orientation. The sequence obtained was submitted to GenBank, and an accession number was obtained (KF 362132).

To optimize the expression condition, a single positive clone was induced with varying concentrations of IPTG, which indicated that induction with 0.5 mM of IPTG for 6 h at 37°C with agitation was optimum for expression of rCu-Zn SOD protein. The recombinant His-tagged Cu-Zn SOD protein was purified under denaturing conditions by nickel affinity chromatography. Elution of recombinant protein occurred in elution buffer containing imidazole between 40 and 150 mM, with the highest elution occurring at an 80 mM concentration of imidazole. The SDS-PAGE analysis revealed the expected recombinant fusion protein of around 18.5 kDa (Fig. 3). The antigenicity of the purified rCu-Zn SOD protein was confirmed by the production of antigen-specific antibodies, as illustrated by the specific interaction between the purified antigen and hyperimmune sera raised in rabbits (Fig. 4). Thus, our findings demonstrate that Cu-Zn SOD from B. abortus 544 is highly immunogenic and could be used as a potential subunit vaccine candidate. The Cu-Zn SOD  gene of Brucella has been studied by different research group as a potential vaccine candidate in various forms like DNA vaccine, recombinant protein based vaccine, live vectored vaccine  and others and it has shown good protective potential against virulent Brucella challenge (Onate et al., 1999; He et al.,2002; Onate et al., 2003; Munoz-Montesino et al., 2004; Onate et al., 2005; Saez et al., 2008; Singha et al., 2008; Saez et al., 2012; Kim et al., 2019; Wang et al., 2023). This recombinant protein obtained  in this study will be evaluated for its efficacy as a vaccine alone and in combination with the rL7/L12 protein (Rajagunalan et al., 2014) in a mouse model in further studies.

Fig. 2. Restriction enzyme digestion of plasmid containing Cu/Zn SOD gene

Lane M: 100 bp plus DNA ladder, Lane 1: RE digested plasmid showing release of Cu-Zn SOD insert, Lane 2: Undigested plasmid

Fig. 3. Purification of recombinant Cu-Zn SOD protein. SDS-PAGE analysis showing the expected molecular weight of the recombinant protein (~18.5 kDa) after purification.

Fig. 4. Western blot showing reaction of rCu/Zn SOD protein with antiserum raised in rabbit.

In conclusion, the sodC gene of Brucella abortus 544 was successfully cloned, expressed, and purified. The recombinant Cu-Zn SOD protein demonstrated promising immunogenicity, supporting its potential as a subunit vaccine candidate.

The recombinant Cu-Zn SOD protein expressed here will be evaluated for its efficacy in laboratory animal models, both as a standalone vaccine and in combination with other Brucella recombinant proteins.