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Development of a filter device for the prevention of aquatic bacterial disease using a single-chain variable fragment (scFv)-conjugated affinity silk.

ABSTRACT: Infectious disease is one of the most serious problems in the aquaculture industry for ornamental or edible fish. This study attempted to develop a new device for preventing an aquatic bacterial disease, ulcer disease, caused by Aeromonas salmonicida (As), using "affinity silk". Affinity silk is a silk protein-containing fibroin L-chain (FibL) fused to the single-chain variable fragment (scFv). It can be easily processed into different formats such as fibers, gels, sponges, or films. A transgenic silkworm that could express a cDNA construct containing FibL fused to an scFv derived from a monoclonal antibody (MAb) against As was successfully generated. An enzyme-linked immunosorbent assay was used to detect As by employing 96-well plates coated with scFv-conjugated affinity silk. As could be captured efficiently by glass wool coated with affinity silk in the column. Furthermore, the air-lift water filter equipped with the affinity silk-coated wool could considerably reduce the concentration of As in water and was estimated to have sufficient ability to trap a lethal dose of As. These findings show that the "affinity silk filter" is a potential device for the prophylaxis of aquatic animal diseases.

SUBMITTER: Prakash H

PROVIDER: S-EPMC9177605 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Development of a filter device for the prevention of aquatic bacterial disease using a single-chain variable fragment (scFv)-conjugated affinity silk.

Prakash Harsha H Sato Mitsuru M Kojima Katsura K Sato Atsushi A Maruyama Shinpei S Nagasawa Takahiro T Nakao Miki M Somamoto Tomonori T

Scientific reports 20220608 1

Infectious disease is one of the most serious problems in the aquaculture industry for ornamental or edible fish. This study attempted to develop a new device for preventing an aquatic bacterial disease, ulcer disease, caused by Aeromonas salmonicida (As), using "affinity silk". Affinity silk is a silk protein-containing fibroin L-chain (FibL) fused to the single-chain variable fragment (scFv). It can be easily processed into different formats such as fibers, gels, sponges, or films. A transgeni ...[more]

PMID: 35676314

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Project description:BackgroundIntimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). Both pathogens are still important causes of diarrhea in children and adults in many developing and industrialized countries. Considering the fact that antibodies are important tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int388-667). In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv).FindingsAnti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into pGEM-T Easy vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. E. coli BL21(DE3)pLys strain was transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv, expressed as inclusion bodies (insoluble fraction), was denatured, purified and submitted to refolding. The protein yield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin, resulting in an absorbance of 0.75 at 492 nm. The immunofluorescence assay showed a strong reactivity with EPEC E2348/69.ConclusionThis study demonstrated that the recombinant anti-intimin antibody obtained is able to recognize the conserved region of intimin (Int388-667) in purified form and the EPEC isolate.

Dataset Information

Development of a filter device for the prevention of aquatic bacterial disease using a single-chain variable fragment (scFv)-conjugated affinity silk.

Publications

Development of a filter device for the prevention of aquatic bacterial disease using a single-chain variable fragment (scFv)-conjugated affinity silk.

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