Food-grade Selection Markers in Lactic Acid Bacteria

Song, He; Gong, Fanghong; Guo, Ya 'nan; Zhang, Dechun

doi:10.5455/pmb.1-1309507875

Cited by 7 publications

(6 citation statements)

References 84 publications

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“…A disadvantage of using live LAB mucosal vaccines is the possibility of introducing genetically altered organisms with antibiotic resistance genes into the environment and host microflora. As a result, auxotrophic mutants and food‐grade selection markers (such as bacteriocin resistance and generation), depending on the existence of these selectable markers on the plasmid of interest, could allay worries about the risk of unchecked release of nucleic acids into the environment by reducing the use of antibiotic resistance markers and harmful substances and potentially improve the applicability on an industrial level or in food products (He et al., 2012 ).…”

Section: Discussionmentioning

confidence: 99%

Brucella abortus antigen omp25 vaccines: Development and targeting based on Lactococcus lactis

Gouran,

Doosti,

Jami

2023

Veterinary Medicine & Sci

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Background Most Brucella infections take place on mucosal membranes. Therefore, creating vaccinations delivered through the mucosa may be crucial for managing brucellosis. Consequently, we assessed the efficacy of a recombinant oral antigen delivery system based on Lactococcus lactis for Brucella abortus omp25 antigen. Method Oral vaccinations with L. lactis transformed with pNZ8148 variants encoding for omp25 (pNZ8148:omp25) and free‐pNZ8148 were administered to mice. On day 30, following immunization in animal groups, anti‐omp25‐specific IgG1 antibodies were assessed by the ELISA test. Additionally, nasal and bronchoalveolar lavages containing omp25‐specific secretory IgA (sIgA) were analysed by ELISA. ELISA test and real‐time PCR were also used to analyse cytokine responses up to 28 days following the last boost. In addition, the protective potential of L. lactis pNZ8148:omp25 vaccines was assessed in BALB/c mice by exposing them to the B. abortus strain. Results Based on the initial screening results, the omp25 protein was identified for immunogenicity because it had the maximum solubility and flexibility and antigenic values of 0.75. The produced plasmid was digested using KpnI and XbaI. By electrophoretic isolation of the digestion fragments at 786 bp, the omp25 gene, the successful production of the recombinant plasmid, was confirmed. Antigen expression at the protein level revealed that the target group generated the 25 kDa‐sized omp25 protein, but there was no protein expression in the control group. Fourteen days after priming, there was a considerable amount of omp25‐specific IgG1 in the sera of mice vaccinated with pNZ8148–Usp45–omp25–L. lactis (p < 0.001 in target groups compared to the phosphate‐buffered saline control group). IFN‐γ and TNF‐α levels were more significant in samples from mice that had been given the pNZ8148–Usp45–omp25–L. lactis and IRBA vaccinations, in samples taken on days 14 and 28, respectively (p < 0.001). The pNZ8148–Usp45–omp25–L. lactis and IRBA immunization groups had significantly greater IL‐4 and IL‐10 transcription levels than the other groups. The spleen portions from the pNZ8148–Usp45–omp25–L. lactis and IRIBA vac group had less extensive spleen injuries, alveolar oedema, lymphocyte infiltration and morphological damage due to the inflammatory process. Conclusion Our study offers a novel method for using the food‐grade, non‐pathogenic and noncommercial bacterium L. lactis as a protein cell factory to produce the novel immunogenic fusion candidate romp25. This method offers an appealing new approach to assessing the cost‐effective, safe, sustainable, simple pilot development of pharmaceutical products.

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Section: Discussionmentioning

confidence: 99%

Brucella abortus antigen omp25 vaccines: Development and targeting based on Lactococcus lactis

Gouran,

Doosti,

Jami

2023

Veterinary Medicine & Sci

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“…In addition to the limitations associated with the engineering of foodgrade plasmids, the lack of a readily selectable marker to allow their transfer is another aspect that often restricts their use. Due to the restrictions associated with using antibiotic markers in food applications, three alternative types of selection have been used [33]. The first of these, involving the use of dominant markers such as bacteriocin production and immunity [34] or heavy metal resistance [35], is relatively straightforward but depends upon the presence of these selectable markers on the plasmid of interest.…”

Section: Discussionmentioning

confidence: 99%

Heterologous expression and activity studying of a nisin resistance gene in <i>Lactobacillus delbrueckii</i> subsp. bulgaricus

Song¹,

Hu²,

Zhang³

et al. 2013

J Exp Integr Med

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Background: Lactic acid bacteria are generally regarded as safe microorganisms and widely used in industry and medicine. We are trying to add additional properties to them by gene engineering. However, the genetically modified bacteria are not acceptable to use in food and medicine due to the presence of antibiotic resistance genes in plasmids. Thus, it is necessary to develop food-grade selection markers. Methods: To evaluate the activity of nisin resistance gene (nsr) cloned from Lactococcus lactis as a food-grade selective marker in transformant bacterial strains, we isolated L.lactis nisin resistant strains from fresh milk, and cloned the nsr gene to Lactobacillus delbrueckii subsp. bulgaricus, then studied the activity of nsr in positive transformant bacterial strains. Results: Six nisin resistant strains were isolated from fresh milk on M17 plates supplemented with nisin at a final concentration of 400 μg/ml. All of these isolates were identified as L.lactis by morphological, physiological, biochemical and 16S rDNA sequence analysis. A pair of primers was designed on the basis of the DNA sequences of reported nsr. PCR amplification was carried out with chromosome and plasmid DNA as templates from these six strains respectively, and an expected PCR product was obtained from one chromosome. After the amplicon was confirmed as nsr gene, it was cloned into E.coli-Lactobacillus shuttle vector pMG36e, resulting in pMG36ensr. The construct was obtained when the recombinant plasmid pMG36e-nsr was electroporated into L.delbrueckii subsp. bulgaricus L6032 competent cells. When the medium contained a maximum of 400 μg/ml nisin, the construct carrying pMG36e-nsr showed the same growth curve as that cultured in medium containing 10 μg/ml erythromycin. Conclusions: The nsr gene was cloned and successfully expressed in L.delbrueckii subsp. bulgaricus L6032. This study suggests that nsr gene could be used as a potential food-grade selective marker to replace antibiotic resistant markers in future.

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“…; He et al . ). Active containment is based on the conditional genetic control of either activation of a compound or repression of an essential gene.…”

Section: Doubtsmentioning

confidence: 97%

Lactic acid bacteria - promising vaccine vectors: possibilities, limitations, doubts

2017

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SummaryGram-positive, nonpathogenic lactic acid bacteria (LAB) are considered to be promising candidates for the development of novel, safe production and delivery systems of heterologous proteins. Recombinant LAB strains were shown to elicit specific systemic and mucosal immune responses against selected antigens. For this reason, this group of bacteria is considered as a potential replacement of classical, often pathogenic, attenuated microbial carriers. Mucosal administration of recombinant LAB, especially via the best explored and universal oral route, offers many advantages in comparison to systemic inoculation, and is attractive from the immunological and practical point of view. Research aimed at designing efficient, mucosally applied vaccines in combination with improved immunization efficiency, monitoring of in vivo antigen production, determination of optimal dose for vaccination, strain selection and characterization is a priority in modern vaccinology. This paper summarizes and organizes the available knowledge on the application of LAB as live oral vaccine vectors. It constitutes a valuable source of general information for researchers interested in mucosal vaccine development and constructing LAB strains with vaccine potential.

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Food-grade Selection Markers in Lactic Acid Bacteria

Cited by 7 publications

References 84 publications

Brucella abortus antigen omp25 vaccines: Development and targeting based on Lactococcus lactis

Brucella abortus antigen omp25 vaccines: Development and targeting based on Lactococcus lactis

Heterologous expression and activity studying of a nisin resistance gene in <i>Lactobacillus delbrueckii</i> subsp. bulgaricus

Lactic acid bacteria - promising vaccine vectors: possibilities, limitations, doubts

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Food-grade Selection Markers in Lactic Acid Bacteria

Cited by 7 publications

References 84 publications

Brucella abortus antigen omp25 vaccines: Development and targeting based on Lactococcus lactis

Brucella abortus antigen omp25 vaccines: Development and targeting based on Lactococcus lactis

Heterologous expression and activity studying of a nisin resistance gene in &lt;i&gt;Lactobacillus delbrueckii&lt;/i&gt; subsp. bulgaricus

Lactic acid bacteria - promising vaccine vectors: possibilities, limitations, doubts

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Heterologous expression and activity studying of a nisin resistance gene in <i>Lactobacillus delbrueckii</i> subsp. bulgaricus