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Characterization of Anti-Citrinin Specific ScFvs Selected from Non-Immunized Mouse Splenocytes by Eukaryotic Ribosome Display.

Cheng H, Chen Y, Yang Y, Chen X, Guo X, Du A - PLoS ONE (2015)

Bottom Line: The three scFvs showed high specificity to CIT and the cross reactivity with other mycotoxins was below 0.01% as determined by indirect competitive ELISA.These specific scFvs offer a potential novel immunoassay method for CIT residues.This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for the selection of scFvs specific to other small molecules using ribosome display.

View Article: PubMed Central - PubMed

Affiliation: Institute of Preventive Veterinary Medicine & Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, China.

ABSTRACT
Single chain variable fragments (scFvs) against citrinin (CIT) were selected from a scFv library constructed from the splenocytes of non-immunized mice by an improved eukaryotic ribosome display technology in this study. Bovine serum albumin (BSA)/ CIT-BSA and ovalbumin (OVA)/ CIT-OVA were used as the antigens to select specific anti-CIT scFvs. Eukaryotic in situ RT-PCR method was used to recover the selected mRNA after every affinity selection. After six rounds of ribosome display, expression vector pTIG-TRX carrying specific scFv DNAs were constructed and transformed into Escherichia coli BL21 (DE3) for protein expression. Thirteen positive clones were selected out of which three (designated 23, 68 and 109) showed high binding activity and specificity to CIT by indirect ELISA, while no clone showed binding activity with carrier proteins. The three scFvs showed high specificity to CIT and the cross reactivity with other mycotoxins was below 0.01% as determined by indirect competitive ELISA. These specific scFvs offer a potential novel immunoassay method for CIT residues. This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for the selection of scFvs specific to other small molecules using ribosome display.

No MeSH data available.


Related in: MedlinePlus

Agarose gel electrophoresis of the amplified DNA fragments of VH, VL, Cκ, GS Linker and assembled scFv library, assembled ribosome display library.A: The amplified DNA fragments of VH and VL. Lane 1: The fragment of VH; Lane 2: The fragment of VL; Lane M: 100bp DNA ladder marker. B: The amplified DNA fragment of Cκ. Lane 1: The fragment of Cκ; Lane M: 100bp DNA ladder marker. C: The amplified DNA fragment of GS Linker. Lane 1: The fragment of GS Linker; Lane M: 100bp DNA ladder marker. D: The amplified DNA fragments of assembled scFv library and ribosome display library. Lane 1: The fragment of assembled scFv library; Lane 2: The fragment of assembled ribosome display library; Lane M: 100bp DNA ladder marker.
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pone.0131482.g005: Agarose gel electrophoresis of the amplified DNA fragments of VH, VL, Cκ, GS Linker and assembled scFv library, assembled ribosome display library.A: The amplified DNA fragments of VH and VL. Lane 1: The fragment of VH; Lane 2: The fragment of VL; Lane M: 100bp DNA ladder marker. B: The amplified DNA fragment of Cκ. Lane 1: The fragment of Cκ; Lane M: 100bp DNA ladder marker. C: The amplified DNA fragment of GS Linker. Lane 1: The fragment of GS Linker; Lane M: 100bp DNA ladder marker. D: The amplified DNA fragments of assembled scFv library and ribosome display library. Lane 1: The fragment of assembled scFv library; Lane 2: The fragment of assembled ribosome display library; Lane M: 100bp DNA ladder marker.

Mentions: The VH and VL fragments of the expected size were amplified (Fig 5). The (Gly4Ser) 3-linker fragment was synthesized with the two ends complementary to the downstream sequence of the VH fragments and the upstream sequence of the VL fragments, respectively (Fig 5). The scFv library, used to construct the ribosome display library, was assembled with the VH, VL, and the (Gly4Ser) 3-linker by SOE-PCR (Fig 5).


Characterization of Anti-Citrinin Specific ScFvs Selected from Non-Immunized Mouse Splenocytes by Eukaryotic Ribosome Display.

Cheng H, Chen Y, Yang Y, Chen X, Guo X, Du A - PLoS ONE (2015)

Agarose gel electrophoresis of the amplified DNA fragments of VH, VL, Cκ, GS Linker and assembled scFv library, assembled ribosome display library.A: The amplified DNA fragments of VH and VL. Lane 1: The fragment of VH; Lane 2: The fragment of VL; Lane M: 100bp DNA ladder marker. B: The amplified DNA fragment of Cκ. Lane 1: The fragment of Cκ; Lane M: 100bp DNA ladder marker. C: The amplified DNA fragment of GS Linker. Lane 1: The fragment of GS Linker; Lane M: 100bp DNA ladder marker. D: The amplified DNA fragments of assembled scFv library and ribosome display library. Lane 1: The fragment of assembled scFv library; Lane 2: The fragment of assembled ribosome display library; Lane M: 100bp DNA ladder marker.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488840&req=5

pone.0131482.g005: Agarose gel electrophoresis of the amplified DNA fragments of VH, VL, Cκ, GS Linker and assembled scFv library, assembled ribosome display library.A: The amplified DNA fragments of VH and VL. Lane 1: The fragment of VH; Lane 2: The fragment of VL; Lane M: 100bp DNA ladder marker. B: The amplified DNA fragment of Cκ. Lane 1: The fragment of Cκ; Lane M: 100bp DNA ladder marker. C: The amplified DNA fragment of GS Linker. Lane 1: The fragment of GS Linker; Lane M: 100bp DNA ladder marker. D: The amplified DNA fragments of assembled scFv library and ribosome display library. Lane 1: The fragment of assembled scFv library; Lane 2: The fragment of assembled ribosome display library; Lane M: 100bp DNA ladder marker.
Mentions: The VH and VL fragments of the expected size were amplified (Fig 5). The (Gly4Ser) 3-linker fragment was synthesized with the two ends complementary to the downstream sequence of the VH fragments and the upstream sequence of the VL fragments, respectively (Fig 5). The scFv library, used to construct the ribosome display library, was assembled with the VH, VL, and the (Gly4Ser) 3-linker by SOE-PCR (Fig 5).

Bottom Line: The three scFvs showed high specificity to CIT and the cross reactivity with other mycotoxins was below 0.01% as determined by indirect competitive ELISA.These specific scFvs offer a potential novel immunoassay method for CIT residues.This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for the selection of scFvs specific to other small molecules using ribosome display.

View Article: PubMed Central - PubMed

Affiliation: Institute of Preventive Veterinary Medicine & Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, China.

ABSTRACT
Single chain variable fragments (scFvs) against citrinin (CIT) were selected from a scFv library constructed from the splenocytes of non-immunized mice by an improved eukaryotic ribosome display technology in this study. Bovine serum albumin (BSA)/ CIT-BSA and ovalbumin (OVA)/ CIT-OVA were used as the antigens to select specific anti-CIT scFvs. Eukaryotic in situ RT-PCR method was used to recover the selected mRNA after every affinity selection. After six rounds of ribosome display, expression vector pTIG-TRX carrying specific scFv DNAs were constructed and transformed into Escherichia coli BL21 (DE3) for protein expression. Thirteen positive clones were selected out of which three (designated 23, 68 and 109) showed high binding activity and specificity to CIT by indirect ELISA, while no clone showed binding activity with carrier proteins. The three scFvs showed high specificity to CIT and the cross reactivity with other mycotoxins was below 0.01% as determined by indirect competitive ELISA. These specific scFvs offer a potential novel immunoassay method for CIT residues. This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for the selection of scFvs specific to other small molecules using ribosome display.

No MeSH data available.


Related in: MedlinePlus