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Maghsood Ahmadi F, Mahboubi A, Hosseini F, Esmaeili D, Hajikhani B. Cloning, expression, and purification of recombinant Enterotoxin B and cholera toxin B fusion protein in Lactobacillus plantarum. mljgoums 2025; 19 (5) :36-39
URL: http://mlj.goums.ac.ir/article-1-1387-en.html
URL: http://mlj.goums.ac.ir/article-1-1387-en.html
Fatemeh Maghsood Ahmadi1 
, Arash Mahboubi2 , Farzaneh Hosseini1 , Davoud Esmaeili3 , Bahareh Hajikhani4
, Arash Mahboubi2 , Farzaneh Hosseini1 , Davoud Esmaeili3 , Bahareh Hajikhani4
1- Department of Microbiology NT.C., Islamic Azad University, Tehran, Iran
2- Department of Pharmaceutics, School of Pharmacy, Shahid Beheshti University of Medical Science, Tehran, Iran; Food Safety Research Center, Shahid Beheshti University of Medical Science, Tehran, Iran ,mahboubi@sbmu.ac.ir
3- Department of Microbiology, Baqiyatallah University of Medical Sciences, Tehran, Iran; Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
4- Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2- Department of Pharmaceutics, School of Pharmacy, Shahid Beheshti University of Medical Science, Tehran, Iran; Food Safety Research Center, Shahid Beheshti University of Medical Science, Tehran, Iran ,
3- Department of Microbiology, Baqiyatallah University of Medical Sciences, Tehran, Iran; Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
4- Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Keywords: Staphylococcus aureus enterotoxin B, Lactobacillus Plantarum, Cloning, Organism, Vaccines, Cholera toxin subunit B
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The expression of the recombinant protein was analyzed by SDS-PAGE and subsequently confirmed via Western blot using an anti-His tag antibody. The Western blot revealed a distinct band at approximately 49 kDa, indicating successful expression of the rSEB-CTXB fusion gene in L. plantarum (Figure 3).
Discussion
The research aims to design an oral vaccine antigen using LAB as a safe carrier, focusing on chimeric proteins to increase both cellular and humoral immune responses (26). Al-Rasoul et al. studied the effectiveness of CS3 antigen combined with LT toxoid, finding that chimeric protein LTB-CstH elicited a stronger immune response (27). This study explores the design of chimeric proteins combating rSEB and CTB antigen proteins, for their immunogenicity and CTB adjuvant properties, following a 2020 study using ctxB (28). This study showed that fusion antigens with CTB offer enhanced stability and longer half-life to preserve the structural integrity of each protein, coding sequences were joined using EAAAK-based linkers to expose linear and conformational epitopes for effective immune recognition (29). Khaloiee et al. used four repeats of this linker to design chimeric proteins targeting three intestinal pathogens, ensuring spatial separation of subunits and proper folding (30). Nazarian et al. used quadruple linker replication to separate chimeric protein subunits, enhancing folding and biological activity (23). Amani et al. showed that EAAAK linkers maintained distinct structures for EspA, intimin, and Tir. Due to rare codons in ctxB and seb genes, all chimeric sequences were codon-optimized for efficient expression in L. plantarum (29).
Codon selection and optimization effectively enhanced expression efficiency, raising the chimeric CAI to 0.8-reflecting improved tRNA availability in L. plantarum. The LAB strains are widely recognized as robust hosts for heterologous protein production due to their resilience in gastrointestinal environments, ability to colonize intestinal tissues, and safe, beneficial characteristics (31,32). The LAB strains have been successfully employed to deliver bioactive molecules, including vaccines, to the gut mucosa (33,34). Prior studies have demonstrated recombinant lactobacilli expressing antigens such as Streptococcus pneumoniae PsaA and tetanus toxin fragments (35). Notably, Guo (2020) utilized L. plantarum to produce and secrete rChIL17B, effectively inhibiting infectious bronchitis virus (IBV) propagation (36).
In a 2020 study by Jianzhong Wang, recombinant L. plantarum NC8 was used to develop a safer and more effective oral rabies vaccine. This strain showed promise as a novel approach for rabies prevention in animals (37). Using safe bacteria such as LAB offer a viable alternative as antigen carriers due to their adjuvant properties and low immunogenicity. The concept of using live bacteria to deliver vaccine antigens dates back to the 1980s. L. plantarum was selected for its stronger innate immune-stimulating capacity compared to species like L. casei and L. lactis. Additionally, a 2007 study by Cortes-Perez demonstrated that nasal immunization with the E7 antigen bound to L. plantarum’s cell wall triggered a robust systemic immune response (38,39).
Conclusion
This study examined the expression of the recombinant rSEB-CTB protein using L. plantarum as the host and pNZ7021 expression vector. The resulting LP-pNZ7021 - SP-rseb-ctxB is suitable for recombinant vaccines against Vibrio cholera and Staphylococcus aureus, and holds promise for immunogenic vaccine applications.
Acknowledgement
We appreciate the support of the Research Council of Shahid Beheshti University.
Funding sources
This study was supported by the Faculty of Pharmacy, Shahid Beheshti University.
Ethical statement
Not applicable.
Conflicts of interest
The authors declare no conflicts of interest.
Author contributions
Each author contributed to the design and conceptualization of the study.
Data availability statement
The research data produced in this work are publicly accessible through PubMed and Scopus.
Full-Text: (45 Views)
Introduction
Staphylococcus aureus is a significant global cause of foodborne illnesses, producing various virulence factors that contribute to its pathogenicity (1-5). Among these, S. aureus enterotoxin B (SEB) is a potent superantigen linked to congenital poisoning, toxic shock, and septic shock syndrome, especially in pregnant women, making it a potential vaccine antigen (6). In many developing countries, cholera remains a leading cause of child morbidity and mortality. Its symptoms are primarily driven by cholera toxin (CT), an 85 kDa AB5-type protein composed of CTA and CTB subunits. The CTB subunit, a non-toxic homopentamer, facilitates cell binding and acts as a mucosal adjuvant, enhancing both humoral and mucosal immunity (7,8). It is a mucosal supplement for oral and nasal vaccines (9), and is a mucosal delivery or carrier system (10). By fusing Staphylococcus aureus enterotoxin with cholera toxin subunit B (CTB), it is hypothesized that CTB enhances the immune response against enterotoxin and enables immunogenicity against both pathogens. This study explored a chimeric construct combining SEB and CTB to develop a dual-target vaccine candidate. Lactic acid bacteria (LAB) were used as a delivery system, capable of directing heterologous proteins to the mucosal immune system. A 2003 study by Boles JW demonstrated successful cloning and expression of a mutant SEB protein in LAB, resulting in immunogenicity in mice (11). LAB have been widely employed for antigen expression and delivery of therapeutic proteins to mucosal tissues (12). Their growing use in mucosal secretion systems is supported by their role in the gut microbiota and their probiotic benefits (13-17).
In addition, to produce minimal immune responses against itself, LAB can induce a high level of systemic and mucosal antibodies against foreign antigens expressed on its surface following absorption by the mucosal immune system (2,18). Unlike the currently used vectors, LAB, due to their probiotic and anti-inflammatory attributes, can stimulate the host immune system and is also considered an inactive microorganism. Among the LAB vectors, lactobacilli are considered a good option due to their stimulating immunity and greater ability to survive in the mucosal layer. Vaccination is the best way to control enterotoxaemia (3), and due to the problems of conventional vaccines in production processes and quality control stages, their production is not recommended (19). Therefore, the use of LAB in vaccine delivery is approved as an effective method. Advantages of recombinant toxin vaccines include lack of toxicity, greater stability, and greater immunogenicity (19-21). Since very few studies have been conducted in this field in Iran and considering the importance of foodborne diseases in developing countries and around the world, this study was designed to use a potential native probiotic Lactobacillus. In this study, we aimed to design a recombinant rseb-ctxB fusion gene vaccine in Lactobacillus and purify the protein. Vaccination with it in future studies could lead to a reduction in the incidence of infectious diseases of the gastrointestinal tract and reduce the use of antibiotics in the country. Therefore, in the present study, the rSEB-CTXB fusion gene was expressed in L. plantarum host in the secretory model using pNZ7021 vector serve as a candidate for the chimeric vaccine.
Methods
Bacterial strains and growth conditions
Lactobacillus plantarum (L. plantarum) 299v. (Purchased from the Pasteur Institute of Iran) and recombinant L. plantarum 299v were grown in M17 (Merck) medium containing 0.5% glucose (GM17) under anaerobic conditions at 30 °C. The cultures were then incubated for 48 hours without shaking (22).
Sequence analysis, segment selection, gene synthesis, and cloning
The ctxB and SEB sequences were obtained from GenBank and stored in FASTA format. A linker repeat (EAAAK) was used to maintain protein structure and prevent dominic interference (23). The rseb variant was obtained after the mutations L45R, Y89A, and Y94A, respectively (24). The Canadian Biometrics Company synthesized and optimized a fusion gene for recombinant chimeric protein, which was then cloned into the pBlueScript II SK (+) vector. Enzyme digestion confirmed the correct synthesis of the desired gene fragments. The pBlueScript II SK (+) and pNZ7021 vectors were digested with SphI and HindIII, and a purified rseb-ctxB gene was ligated to the pNZ7021 vector. This vector is used to express genes in LAB bacteria, overexpress homologous and heterogeneous genes, and perform metabolic engineering. The recombinant expression vector pNZ7021-Sp-rSeb-ctxB was transformed into Electrocompetent L. plantarum to express extracellular rSEB-CTB in L. plantarum.
Preparation of electrocompetent cells
The overnight L. plantarum culture was diluted in fresh MRS medium and incubated for 4 hours at 30 °C. The pellets were washed twice with MgCl2, sucrose, and glycerol, and then resuspended in the same solution and kept on ice.
Electrification of cells
Two µL pNZ7021 vector was added to competent cells, homogenized, and transferred to pre-cooled cuvettes. The mixture was transferred into cuvettes and electroporated in a Gene Pulser device (25). The bacteria were cultured on MARS agar medium, and chloramphenicol-resistant colonies were obtained. Clones were examined using PCR and primers synthesized by Sinaclone. The results confirmed the presence of a 976-bp amplified fragment in the PCR colonies.
Purification of recombinant proteins using a nickel resin chromatography column
The recombinant protein sequence was purified using a nickel column (Ni-NTA) with a histidine sequence affinity. The process involved pouring prepared nickel-resin into an empty column, regenerating it with DBB, and adding the extracted protein sample. The column was washed with DWB denaturing Wash Buffer, NWB buffer, and imidazole buffers. The process was repeated, and the collected samples were stored at room temperature.
Western blot analysis
The protein extract was prepared from recombinant L. plantarum culture medium. It was centrifuged, precipitated, and then centrifuged again. The supernatant proteins were then mixed with NaOH, PMSF, and DTT-LB (22). Proteins from LP-pNZ7021-Sp-rseb-ctxB and L. plantarum were analyzed using SDS-PAGE and Western blotting, with anti-His tag antibody detection for target protein presence.
Results
Sequence analysis and segment selection
The gene sequences for ctxB (372 bp) and SEB (798 bp), as referenced under GenBank accession number AB462486.1, were retrieved for this study. These segments encode a total of 390 amino acids-124 from CTB and 266 from SEB. To create a synthetic antigenic construct (SC), the two antigenic fragments were fused using specific linker sequences. In this design, repetitive linkers such as AEAAAKEAAAKEAAAKEAAAKA were employed to ensure proper structural flexibility and separation between domains.
Optimized gene constructs
The gene constructs were optimized for expression in Lactobacillus plantarum 299v, resulting in a codon adaptation index (CAI) of 0.8. Codon usage was appropriately modified, leading to a 33.5% increase in GC content and a reduction in destabilizing motifs and repetitive sequences. Enzymatic digestion was performed to extract the desired gene fragments from pBlueScript II SK vector (+). Restriction enzyme digestion using KpnI and EcoRI was carried out on the pBlueScript II SK (+) plasmid by Canadian Biometrics Company, confirming the accurate synthesis and insertion of the target gene fragments (Figure 1).
Staphylococcus aureus is a significant global cause of foodborne illnesses, producing various virulence factors that contribute to its pathogenicity (1-5). Among these, S. aureus enterotoxin B (SEB) is a potent superantigen linked to congenital poisoning, toxic shock, and septic shock syndrome, especially in pregnant women, making it a potential vaccine antigen (6). In many developing countries, cholera remains a leading cause of child morbidity and mortality. Its symptoms are primarily driven by cholera toxin (CT), an 85 kDa AB5-type protein composed of CTA and CTB subunits. The CTB subunit, a non-toxic homopentamer, facilitates cell binding and acts as a mucosal adjuvant, enhancing both humoral and mucosal immunity (7,8). It is a mucosal supplement for oral and nasal vaccines (9), and is a mucosal delivery or carrier system (10). By fusing Staphylococcus aureus enterotoxin with cholera toxin subunit B (CTB), it is hypothesized that CTB enhances the immune response against enterotoxin and enables immunogenicity against both pathogens. This study explored a chimeric construct combining SEB and CTB to develop a dual-target vaccine candidate. Lactic acid bacteria (LAB) were used as a delivery system, capable of directing heterologous proteins to the mucosal immune system. A 2003 study by Boles JW demonstrated successful cloning and expression of a mutant SEB protein in LAB, resulting in immunogenicity in mice (11). LAB have been widely employed for antigen expression and delivery of therapeutic proteins to mucosal tissues (12). Their growing use in mucosal secretion systems is supported by their role in the gut microbiota and their probiotic benefits (13-17).
In addition, to produce minimal immune responses against itself, LAB can induce a high level of systemic and mucosal antibodies against foreign antigens expressed on its surface following absorption by the mucosal immune system (2,18). Unlike the currently used vectors, LAB, due to their probiotic and anti-inflammatory attributes, can stimulate the host immune system and is also considered an inactive microorganism. Among the LAB vectors, lactobacilli are considered a good option due to their stimulating immunity and greater ability to survive in the mucosal layer. Vaccination is the best way to control enterotoxaemia (3), and due to the problems of conventional vaccines in production processes and quality control stages, their production is not recommended (19). Therefore, the use of LAB in vaccine delivery is approved as an effective method. Advantages of recombinant toxin vaccines include lack of toxicity, greater stability, and greater immunogenicity (19-21). Since very few studies have been conducted in this field in Iran and considering the importance of foodborne diseases in developing countries and around the world, this study was designed to use a potential native probiotic Lactobacillus. In this study, we aimed to design a recombinant rseb-ctxB fusion gene vaccine in Lactobacillus and purify the protein. Vaccination with it in future studies could lead to a reduction in the incidence of infectious diseases of the gastrointestinal tract and reduce the use of antibiotics in the country. Therefore, in the present study, the rSEB-CTXB fusion gene was expressed in L. plantarum host in the secretory model using pNZ7021 vector serve as a candidate for the chimeric vaccine.
Methods
Bacterial strains and growth conditions
Lactobacillus plantarum (L. plantarum) 299v. (Purchased from the Pasteur Institute of Iran) and recombinant L. plantarum 299v were grown in M17 (Merck) medium containing 0.5% glucose (GM17) under anaerobic conditions at 30 °C. The cultures were then incubated for 48 hours without shaking (22).
Sequence analysis, segment selection, gene synthesis, and cloning
The ctxB and SEB sequences were obtained from GenBank and stored in FASTA format. A linker repeat (EAAAK) was used to maintain protein structure and prevent dominic interference (23). The rseb variant was obtained after the mutations L45R, Y89A, and Y94A, respectively (24). The Canadian Biometrics Company synthesized and optimized a fusion gene for recombinant chimeric protein, which was then cloned into the pBlueScript II SK (+) vector. Enzyme digestion confirmed the correct synthesis of the desired gene fragments. The pBlueScript II SK (+) and pNZ7021 vectors were digested with SphI and HindIII, and a purified rseb-ctxB gene was ligated to the pNZ7021 vector. This vector is used to express genes in LAB bacteria, overexpress homologous and heterogeneous genes, and perform metabolic engineering. The recombinant expression vector pNZ7021-Sp-rSeb-ctxB was transformed into Electrocompetent L. plantarum to express extracellular rSEB-CTB in L. plantarum.
Preparation of electrocompetent cells
The overnight L. plantarum culture was diluted in fresh MRS medium and incubated for 4 hours at 30 °C. The pellets were washed twice with MgCl2, sucrose, and glycerol, and then resuspended in the same solution and kept on ice.
Electrification of cells
Two µL pNZ7021 vector was added to competent cells, homogenized, and transferred to pre-cooled cuvettes. The mixture was transferred into cuvettes and electroporated in a Gene Pulser device (25). The bacteria were cultured on MARS agar medium, and chloramphenicol-resistant colonies were obtained. Clones were examined using PCR and primers synthesized by Sinaclone. The results confirmed the presence of a 976-bp amplified fragment in the PCR colonies.
Purification of recombinant proteins using a nickel resin chromatography column
The recombinant protein sequence was purified using a nickel column (Ni-NTA) with a histidine sequence affinity. The process involved pouring prepared nickel-resin into an empty column, regenerating it with DBB, and adding the extracted protein sample. The column was washed with DWB denaturing Wash Buffer, NWB buffer, and imidazole buffers. The process was repeated, and the collected samples were stored at room temperature.
Western blot analysis
The protein extract was prepared from recombinant L. plantarum culture medium. It was centrifuged, precipitated, and then centrifuged again. The supernatant proteins were then mixed with NaOH, PMSF, and DTT-LB (22). Proteins from LP-pNZ7021-Sp-rseb-ctxB and L. plantarum were analyzed using SDS-PAGE and Western blotting, with anti-His tag antibody detection for target protein presence.
Results
Sequence analysis and segment selection
The gene sequences for ctxB (372 bp) and SEB (798 bp), as referenced under GenBank accession number AB462486.1, were retrieved for this study. These segments encode a total of 390 amino acids-124 from CTB and 266 from SEB. To create a synthetic antigenic construct (SC), the two antigenic fragments were fused using specific linker sequences. In this design, repetitive linkers such as AEAAAKEAAAKEAAAKEAAAKA were employed to ensure proper structural flexibility and separation between domains.
Optimized gene constructs
The gene constructs were optimized for expression in Lactobacillus plantarum 299v, resulting in a codon adaptation index (CAI) of 0.8. Codon usage was appropriately modified, leading to a 33.5% increase in GC content and a reduction in destabilizing motifs and repetitive sequences. Enzymatic digestion was performed to extract the desired gene fragments from pBlueScript II SK vector (+). Restriction enzyme digestion using KpnI and EcoRI was carried out on the pBlueScript II SK (+) plasmid by Canadian Biometrics Company, confirming the accurate synthesis and insertion of the target gene fragments (Figure 1).
.PNG) Figure 1. Enzymatic digestion results show successful synthesis of the target gene fragments, confirmed by the appearance of a 1,500 bp band on agarose gel electrophoresis after restriction enzyme digestion. The presence of the recombinant expression vector in L. plantarum was confirmed |
.PNG) Figure 2. Confirmation of transformation by colony PCR: Detection of a band with a molecular weight of 976 bp on agarose gel electrophoresis. From left to right a. The desired band b. Marker 1kb DM3100. c. Negative control. |
The expression of the recombinant protein was analyzed by SDS-PAGE and subsequently confirmed via Western blot using an anti-His tag antibody. The Western blot revealed a distinct band at approximately 49 kDa, indicating successful expression of the rSEB-CTXB fusion gene in L. plantarum (Figure 3).
.PNG) Figure 3. Shows the detection of the purified rSEB-CTB fusion protein by SDS-PAGE method and confirmation of recombinant protein by Western blotting method using an anti-His tag. A) The rSEB-CTB fusion protein, with a molecular weight of 49 kDa, was detected after purification by a nickel column using SDS-PAGE in the supernatant of recombinant L. plantarum. a: Marker, b: Recombinant protein purified by elution buffer with imidazole 350 mM. B) The expression of recombinant protein purified from L.P-pNZ 7021-SP_rseb-ctxB was confirmed by Western blotting. a: Band 49 kDa, b: Negative control, c: PM1700 protein marker. |
Discussion
The research aims to design an oral vaccine antigen using LAB as a safe carrier, focusing on chimeric proteins to increase both cellular and humoral immune responses (26). Al-Rasoul et al. studied the effectiveness of CS3 antigen combined with LT toxoid, finding that chimeric protein LTB-CstH elicited a stronger immune response (27). This study explores the design of chimeric proteins combating rSEB and CTB antigen proteins, for their immunogenicity and CTB adjuvant properties, following a 2020 study using ctxB (28). This study showed that fusion antigens with CTB offer enhanced stability and longer half-life to preserve the structural integrity of each protein, coding sequences were joined using EAAAK-based linkers to expose linear and conformational epitopes for effective immune recognition (29). Khaloiee et al. used four repeats of this linker to design chimeric proteins targeting three intestinal pathogens, ensuring spatial separation of subunits and proper folding (30). Nazarian et al. used quadruple linker replication to separate chimeric protein subunits, enhancing folding and biological activity (23). Amani et al. showed that EAAAK linkers maintained distinct structures for EspA, intimin, and Tir. Due to rare codons in ctxB and seb genes, all chimeric sequences were codon-optimized for efficient expression in L. plantarum (29).
Codon selection and optimization effectively enhanced expression efficiency, raising the chimeric CAI to 0.8-reflecting improved tRNA availability in L. plantarum. The LAB strains are widely recognized as robust hosts for heterologous protein production due to their resilience in gastrointestinal environments, ability to colonize intestinal tissues, and safe, beneficial characteristics (31,32). The LAB strains have been successfully employed to deliver bioactive molecules, including vaccines, to the gut mucosa (33,34). Prior studies have demonstrated recombinant lactobacilli expressing antigens such as Streptococcus pneumoniae PsaA and tetanus toxin fragments (35). Notably, Guo (2020) utilized L. plantarum to produce and secrete rChIL17B, effectively inhibiting infectious bronchitis virus (IBV) propagation (36).
In a 2020 study by Jianzhong Wang, recombinant L. plantarum NC8 was used to develop a safer and more effective oral rabies vaccine. This strain showed promise as a novel approach for rabies prevention in animals (37). Using safe bacteria such as LAB offer a viable alternative as antigen carriers due to their adjuvant properties and low immunogenicity. The concept of using live bacteria to deliver vaccine antigens dates back to the 1980s. L. plantarum was selected for its stronger innate immune-stimulating capacity compared to species like L. casei and L. lactis. Additionally, a 2007 study by Cortes-Perez demonstrated that nasal immunization with the E7 antigen bound to L. plantarum’s cell wall triggered a robust systemic immune response (38,39).
Conclusion
This study examined the expression of the recombinant rSEB-CTB protein using L. plantarum as the host and pNZ7021 expression vector. The resulting LP-pNZ7021 - SP-rseb-ctxB is suitable for recombinant vaccines against Vibrio cholera and Staphylococcus aureus, and holds promise for immunogenic vaccine applications.
Acknowledgement
We appreciate the support of the Research Council of Shahid Beheshti University.
Funding sources
This study was supported by the Faculty of Pharmacy, Shahid Beheshti University.
Ethical statement
Not applicable.
Conflicts of interest
The authors declare no conflicts of interest.
Author contributions
Each author contributed to the design and conceptualization of the study.
Data availability statement
The research data produced in this work are publicly accessible through PubMed and Scopus.
Research Article: Research Article |
Subject:
Microbiology
Received: 2024/05/4 | Accepted: 2024/11/16 | Published: 2025/12/3 | ePublished: 2025/12/3
Received: 2024/05/4 | Accepted: 2024/11/16 | Published: 2025/12/3 | ePublished: 2025/12/3
References
1. Kadariya J, Smith TC, Thapaliya D. Staphylococcus aureus and staphylococcal food-borne disease: an ongoing challenge in public health. Biomed Res Int. 2014:2014:827965. [View at Publisher] [DOI] [PMID] [Google Scholar]
2. Adachi K, Kawana K, Yokoyama T, Fujii T, Tomio A, Miura S, et al. Oral immunization with a Lactobacillus casei vaccine expressing human papillomavirus (HPV) type 16 E7 is an effective strategy to induce mucosal cytotoxic lymphocytes against HPV16 E7. Vaccine. 2010;28(16):2810-7. [View at Publisher] [DOI] [PMID] [Google Scholar]
3. Milach A, de los Santos JRG, Turnes CG, Moreira ÂN, de Assis RA, Salvarani FM, et al. Production and characterization of Clostridium perfringens recombinant β toxoid. Anaerobe. 2012;18(3):363-5. [View at Publisher] [DOI] [PMID] [Google Scholar]
4. Liu C, Shi C, Li M, Wang M, Ma C, Wang Z. Rapid and simple detection of viable foodborne pathogen Staphylococcus aureus. Front Chem.2019;7:124. [View at Publisher] [DOI] [PMID] [Google Scholar]
5. Sergelidis D, Angelidis AS. Methicillin‐resistant Staphylococcus aureus: a controversial food‐borne pathogen. Lett Appl Microbiol. 2017;64(6):409-18. [View at Publisher] [DOI] [PMID] [Google Scholar]
6. Bae JS, Da F, Liu R, He L, Lv H, Fisher EL, et al. Contribution of Staphylococcal Enterotoxin B to Staphylococcus aureus Systemic Infection. The J Infect Dis. 2021;223(10):1766-75. [View at Publisher] [DOI] [PMID] [Google Scholar]
7. Mathiesen G, Sveen A, Brurberg MB, Fredriksen L, Axelsson L, Eijsink VG. Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1. BMC Genomics. 2009;10:425. [View at Publisher] [DOI] [PMID] [Google Scholar]
8. Jia S, Huang X, Li H, Zheng D, Wang L, Qiao X, et al. Immunogenicity evaluation of recombinant Lactobacillus casei W56 expressing bovine viral diarrhea virus E2 protein in conjunction with cholera toxin B subunit as an adjuvant. Microb Cell Fact. 2020;19(1):186. [View at Publisher] [DOI] [PMID] [Google Scholar]
9. Ferreira DM, Darrieux M, Oliveira MLS, Leite LC, Miyaji EN. Optimized immune response elicited by a DNA vaccine expressing pneumococcal surface protein a is characterized by a balanced immunoglobulin G1 (IgG1)/IgG2a ratio and proinflammatory cytokine production. Clin Vaccine Immunol. 2008;15(3):499-505. [View at Publisher] [DOI] [PMID] [Google Scholar]
10. Souod N, Kargar M, Hoseini MH, Jafarinia M. Fusion-expressed CtxB-TcpA-C-CPE improves both systemic and mucosal humoral and T-cell responses against cholera in mice. Microb Pathog. 2021;157:104978. [View at Publisher] [DOI] [PMID] [Google Scholar]
11. Boles JW, Pitt MLM, LeClaire RD, Gibbs PH, Torres E, Dyas B, et al. Generation of protective immunity by inactivated recombinant staphylococcal enterotoxin B vaccine in nonhuman primates and identification of correlates of immunity. Clin Immunol. 2003;108(1):51-9. [View at Publisher] [DOI] [PMID] [Google Scholar]
12. Bermúdez-Humarán LG, Kharrat P, Chatel J-M, Langella P. Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Microb Cell Fact. 2011;10(1):1-10. [View at Publisher] [DOI] [PMID] [Google Scholar]
13. Allain T, Mansour NM, Bahr MM, Martin R, Florent I, Langella P, et al. A new lactobacilli in vivo expression system for the production and delivery of heterologous proteins at mucosal surfaces. FEMS Microbiol Lett. 2016;363(13):fnw117. [View at Publisher] [DOI] [PMID] [Google Scholar]
14. Ljungh A, Wadstrom T. Lactic acid bacteria as probiotics. Curr Issues Intest Microbiol. 2006;7(2):73-90. [View at Publisher] [PMID] [Google Scholar]
15. Wyszyńska A, Kobierecka P, Bardowski J, Jagusztyn-Krynicka EK. Lactic acid bacteria-20 years exploring their potential as live vectors for mucosal vaccination. Appl Microbiol Biotechnol. 2015;99(7):2967-77. [View at Publisher] [DOI] [PMID] [Google Scholar]
16. Bron PA, Kleerebezem M. Lactic acid bacteria for delivery of endogenous or engineered therapeutic molecules. Front Microbiol. 2018;9:1821. [View at Publisher] [DOI] [PMID] [Google Scholar]
17. Israr B, Kim J, Anam S, Anjum F. Lactic Acid Bacteria as Vectors: A Novel Approach for Mucosal Vaccine Delivery. J Clin Cell. 2018. [View at Publisher] [DOI] [Google Scholar]
18. Dadar M, Shahali Y, Mojgani N. Probiotic bacteria as a functional delivery vehicle for the development of live oral vaccines. In: Mojgani N, Dadar M, editors. Probiotic bacteria and postbiotic metabolites: role in animal and human health. Singapore: Springer; 2021. p. 319-35 [View at Publisher] [DOI] [Google Scholar]
19. Nijland R, Lindner C, van Hartskamp M, Hamoen LW, Kuipers OP. Heterologous production and secretion of Clostridium perfringens beta-toxoid in closely related Gram-positive hosts. J Biotechnol. 2007;127(3):361-72. [View at Publisher] [DOI] [PMID] [Google Scholar]
20. Salvarani FM, Conceicão FR, Cunha CE, Moreira GM, Pires PS, Silva RO, et al. Vaccination with recombinant Clostridium perfringens toxoids α and β promotes elevated antepartum and passive humoral immunity in swine. Vaccine. 2013;31(38):4152-5. [View at Publisher] [DOI] [PMID] [Google Scholar]
21. Plavec TV, Berlec A. Engineering of lactic acid bacteria for delivery of therapeutic proteins and peptides. Appl Microbiol Biotechnol. 2019;103(5):2053-66. [View at Publisher] [DOI] [PMID] [Google Scholar]
22. Asensi GF, de Sales NFF, Dutra FF, Feijó DF, Bozza MT, Ulrich RG, et al. Oral immunization with Lactococcus lactis secreting attenuated recombinant staphylococcal enterotoxin B induces a protective immune response in a murine model. Microb Cell Fact. 2013;12:32. [View at Publisher] [DOI] [PMID] [Google Scholar]
23. Nazarian S, Gargari SLM, Rasooli I, Amani J, Bagheri S, Alerasool M. An in silico chimeric multi subunit vaccine targeting virulence factors of enterotoxigenic Escherichia coli (ETEC) with its bacterial inbuilt adjuvant. J Microbiol Methods. 2012;90(1):36-45. [View at Publisher] [DOI] [PMID] [Google Scholar]
24. Ulrich RG, Olson MA, Bavari S. Development of engineered vaccines effective against structurally related bacterial superantigens. Vaccine. 1998;16(19):1857-64. [View at Publisher] [DOI] [PMID] [Google Scholar]
25. Hongying F, Xianbo W, Fang Y, Yang B, Beiguo L. Oral immunization with recombinant Lactobacillus acidophilus expressing the adhesin Hp0410 of Helicobacter pylori induces mucosal and systemic immune responses. Clin Vaccine Immunol. 2014;21(2):126-32. [View at Publisher] [DOI] [PMID] [Google Scholar]
26. Berzofsky JA, Ahlers JD, Belyakov IM. Strategies for designing and optimizing new generation vaccines. Nat Rev Immunol. 2001;1(3):209-19. [View at Publisher] [DOI] [PMID] [Google Scholar]
27. Alerasol M, Gargari SLM, Nazarian S, Bagheri S. Immunogenicity of a fusion protein comprising coli surface antigen 3 and labile B subunit of enterotoxigenic Escherichia coli. Iran Biomed J. 2014;18(4):212-8. [View at Publisher] [DOI] [PMID] [Google Scholar]
28. Jafari D, Malih S, Gomari MM, Safari M, Jafari R, Farajollahi MM. Designing a chimeric subunit vaccine for influenza virus, based on HA2, M2e and CTxB: a bioinformatics study. BMC Mol Cell Biol. 2020;21(1):89. [View at Publisher] [DOI] [PMID] [Google Scholar]
29. Amani J, Salmanian AH, Rafati S, Mousavi SL. Immunogenic properties of chimeric protein from espA, eae and tir genes of Escherichia coli O157: H7. Vaccine. 2010;28(42):6923-9. [View at Publisher] [DOI] [PMID] [Google Scholar]
30. Khaloiee F, Pourfarzam P, Rasooli I, Amani J, Nazarian S, Mousavi SL. In silico analysis of chimeric recombinant immunogen against three diarrhea causing bacteria. Journal of Cell and Molecular Research. 2013;5(2):65-74. [View at Publisher] [Google Scholar]
31. Sørvig E, Mathiesen G, Naterstad K, Eijsink VG, Axelsson L. High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors. Microbiology. 2005;151(7):2439-49. [View at Publisher] [DOI] [PMID] [Google Scholar]
32. Szatraj K, Szczepankowska AK, Chmielewska‐Jeznach M. Lactic acid bacteria-promising vaccine vectors: possibilities, limitations, doubts. J Appl Microbiol. 2017;123(2):325-39. [View at Publisher] [DOI] [PMID] [Google Scholar]
33. Mathiesen G, Sveen A, Piard JC, Axelsson L, Eijsink VGH. Heterologous protein secretion by Lactobacillus plantarum using homologous signal peptides. J Appl Microbiol. 2008;105(1):215-26. [View at Publisher] [DOI] [PMID] [Google Scholar]
34. Oliveira MLS, Monedero V, Miyaji EN, Leite LCC, Lee Ho P, Pérez-Martínez G. Expression of Streptococcus pneumoniae antigens, PsaA (pneumococcal surface antigen A) and PspA (pneumococcal surface protein A) by Lactobacillus casei. FEMS Microbiol Lett. 2003;227(1):25-31. [View at Publisher] [DOI] [PMID] [Google Scholar]
35. Reveneau N, Geoffroy M-C, Locht C, Chagnaud P, Mercenier A. Comparison of the immune responses induced by local immunizations with recombinant Lactobacillus plantarum producing tetanus toxin fragment C in different cellular locations. Vaccine. 2002;20(13-14):1769-77. [View at Publisher] [DOI] [PMID] [Google Scholar]
36. Guo S, Peng J, Xiao Y, Liu Y, Hao W, Yang X, et al. The Construction and Immunoadjuvant Activities of the Oral Interleukin-17B Expressed by Lactobacillus plantarum NC8 Strain in the Infectious Bronchitis Virus Vaccination of Chickens. Vaccines. 2020;8(2):282. [View at Publisher] [DOI] [PMID] [Google Scholar]
37. Wang J, Jiang H, Yang R, Zhang S, Zhao W, Hu J, et al. Construction and evaluation of recombinant Lactobacillus plantarum NC8 delivering one single or two copies of G protein fused with a DC-targeting peptide (DCpep) as novel oral rabies vaccine. Vet Microbiol. 2020;251:108906. [View at Publisher] [DOI] [PMID] [Google Scholar]
38. Cortes-Perez NG, Lefèvre F, Corthier G, Adel-Patient K, Langella P, Bermúdez-Humarán LG. Influence of the route of immunization and the nature of the bacterial vector on immunogenicity of mucosal vaccines based on lactic acid bacteria. Vaccine. 2007;25(36):6581-8. [View at Publisher] [DOI] [PMID] [Google Scholar]
39. Formal SB, Baron LS, Kopecko DJ, Washington O, Powell C, Life C. Construction of a potential bivalent vaccine strain: introduction of Shigella sonnei form I antigen genes into the galE Salmonella typhi Ty21a typhoid vaccine strain. Infect Immun. 1981;34(3):746-50. [View at Publisher] [DOI] [PMID] [Google Scholar]
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