Development of Lactobacillus paracasei harboring nucleic acid-hydrolyzing 3D8 scFv as a preventive probiotic against murine norovirus infection

Hoang, Phuong Mai; Cho, Seungchan; Kim, Kee Eun; Byun, Sung June; Lee, Taek-Kyun; Lee, Sukchan

doi:10.1007/s00253-014-6257-7

Cited by 38 publications

(33 citation statements)

References 40 publications

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“…Although we did not intend for the CEFs to express such low levels of 3D8 scFv protein, 3D8 scFv anti-viral activity in transgenic CEFs was detectable. Recently, we have expanded our research towards investigating the role of 3D8 through control of anti-viral effect against H1N1 influenza virus and MNV 22, 23 . Thus, we suppose that low level of 3D8 scFv expression could alleviate initial stage of viral propagation.…”

Section: Discussionmentioning

confidence: 99%

Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission

Byun

Yuk

Jang

et al. 2017

Sci Rep

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The 3D8 single chain variable fragment (scFv) is a mini-antibody that causes unusual sequence-independent nuclease activity against all types of nucleic acids. We used recombinant lentiviruses to generate transgenic chickens expressing the 3D8 scFv gene under the control of the chicken β-actin promoter. From 420 injected embryos, 200 chicks (G0) hatched and were screened for the 3D8 scFv using PCR, and 15 chicks were identified as transgenic birds expressing the transgene in their semen. The G0 founder birds were mated with wild-type hens to produce seven transgenic chicks (G1). 3D8 scFv expression in the chicken embryonic fibroblasts (CEFs) was verified by RT-PCR and Western blot analysis. Immunofluorescence staining for 3D8 scFv in the CEFs revealed that the 3D8 scFv protein was primarily cytosolic. To identify 3D8 scFv anti-viral activity, wild-type and two transgenic CEF lines were infected with H9N2 avian influenza virus (AIV). We selected one line of transgenic chickens that exhibited the lowest number of plaque-forming units to be challenged with H9N2 virus. The challenge experiment revealed that contact exposed transgenic chickens expressing 3D8 scFv exhibited suppressed viral shedding. This results suggest that the transgenic chickens developed in this study could be useful for controlling potential within-flock AIV transmission.

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Section: Discussionmentioning

confidence: 99%

Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission

Byun

Yuk

Jang

et al. 2017

Sci Rep

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“…Lactobacillus spp. were used to secrete 6 µg L −1 of Guy’s 13 scFv against a streptococcal adhesion molecule [69], 410 µg L −1 of codon-optimized antiviral 3D8 scFv [70] and 1 mg L −1 of an of llama heavy-chain (VHH) antibody fragment against rotavirus [71]. …”

Section: Discussionmentioning

confidence: 99%

Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains

2017

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BackgroundDifferent strains of the genus Bacillus are versatile candidates for the industrial production and secretion of heterologous proteins. They can be cultivated quite easily, show high growth rates and are usually non-pathogenic and free of endo- and exotoxins. They have the ability to secrete proteins with high efficiency into the growth medium, which allows cost-effective downstream purification processing. Some of the most interesting and challenging heterologous proteins are recombinant antibodies and antibody fragments. They are important and suitable tools in medical research for analytics, diagnostics and therapy. The smallest conventional antibody fragment with high-affinity binding to an antigen is the single-chain fragment variable (scFv). Here, different strains of the genus Bacillus were investigated using diverse cultivation systems for their suitability to produce and secret a recombinant scFv.ResultsExtracellular production of lysozyme-specific scFv D1.3 was realized by constructing a plasmid with a xylose-inducible promoter optimized for Bacillus megaterium and the D1.3scFv gene fused to the coding sequence of the LipA signal peptide from B. megaterium. Functional scFv was successfully secreted with B. megaterium MS941, Bacillus licheniformis MW3 and the three Bacillus subtilis strains 168, DB431 and WB800N differing in the number of produced proteases. Starting with shake flasks (150 mL), the bioprocess was scaled down to microtiter plates (1250 µL) as well as scaled up to laboratory-scale bioreactors (2 L). The highest extracellular concentration of D1.3 scFv (130 mg L−1) and highest space–time-yield (8 mg L−1 h−1) were accomplished with B. subtilis WB800N, a strain deficient in eight proteases. These results were reproduced by the production and secretion of a recombinant penicillin G acylase (Pac).ConclusionsThe genus Bacillus provides high potential microbial host systems for the secretion of challenging heterologous proteins like antibody fragments and large proteins at high titers. In this study, the highest extracellular concentration and space–time-yield of a recombinant antibody fragment for a Gram-positive bacterium so far was achieved. The successful interspecies use of the here-designed plasmid originally optimized for B. megaterium was demonstrated by two examples, an antibody fragment and a penicillin G acylase in up to five different Bacillus strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0625-9) contains supplementary material, which is available to authorized users.

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“…Category S (function unknown, 6.57%) was also abundant. with an LDH (lactose dehydrogenase) constitutive promoter [9]. Then we used the chloramphenicol resistant gene to measure the survival rate and retention time of the administered Lactobacillus, as shown in Fig.…”

Section: Characterization Of a Novel Strain Using High-throughput Seqmentioning

confidence: 99%

“…In our previous study, we found that, after administration of transgenic Lactobacillus paracasei ATCC 334 expressing the 3D8 scFv (single chain variable fragment) protein to mice, the population of Helicobacter species in the intestine decreased, and the population of Pediococcus acidilactici (a probiotic) increased [8]. In addition, transgenic L. paracasei ATCC 334 showed antiviral efficacy against murine norovirus (MNV) [9]. However, the cheese-derived Lactobacillus is expected to have relatively short persistence and survival in the intestine, which can weaken its probiotic effects.…”

Section: Introductionmentioning

confidence: 99%

In vivo and in silico characterization of Lactobacillus reuteri SKKU-OGDONS-01, a potential probiotic from chicken intestine

Kim¹,

Cho²,

Lee³

et al. 2020

Preprint

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Background: Lactobacillus reuteri SKKU-OGDONS-01 was isolated from chicken intestines for further development as an antiviral feed additive. This study aimed to investigate probiotic properties of chicken isolates in mice model and in silico analysis.Results: Compared to known probiotics, Lactobacillus paracasei ATCC 334, Lactobacillus reuteri SKKU-OGDONS-01 showed immune-boosting effects despite short persistence in the mice intestine. Especially, the expression levels of IFN-β and IFN-γ were increased 4 and 40 times higher than those of the control mice. In proportion to the immune-boosting effects elicited by chicken isolates, the antiviral efficacy against murine norovirus (MNV) was also remarkable. For the purpose of evaluating the potential for development as feed additives, the expression levels of probiotic markers such as long-term acid adaptation, stress response, and adhesion-related proteins were investigated using in silico method, and the results showed that these proteins were expressed at high levels in chicken isolate. Conclusion: Our study demonstrated that chicken isolate, Lactobacillus reuteri SKKU-OGDONS-01 can also elicit high probiotic properties in mice even though it originated in chicken. We expect that this chicken isolate will be able to induce much higher probiotic activity in chickens to develop feed additives for poultry.Keywords: Lactobacillus reuteri SKKU-OGDONS-01, probiotics, antiviral efficacy, probiotic marker, cytokine

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Development of Lactobacillus paracasei harboring nucleic acid-hydrolyzing 3D8 scFv as a preventive probiotic against murine norovirus infection

Cited by 38 publications

References 40 publications

Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission

Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission

Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains

In vivo and in silico characterization of Lactobacillus reuteri SKKU-OGDONS-01, a potential probiotic from chicken intestine

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