Dataset Information

The 3D8 single chain variable fragment protein suppresses Newcastle disease virus transmission in transgenic chickens.

ABSTRACT:

Background

The 3D8 single chain variable fragment (scFv) is a mini-antibody sequence that exhibits independent nuclease activity against all types of nucleic acids. In this research, crossing a 3D8 scFv G1 transgenic rooster with wild-type hens produced 3D8 scFv G₂ transgenic chickens to evaluate suppression of viral transmission.

Result

The transgenic chickens were identified using genomic PCR and immunohistochemistry. To evaluate Newcastle disease virus (NDV) protection conferred by 3D8 scFv expression, transgenic, non-transgenic, and specific pathogen-free (SPF) chickens were challenged with virulent NDV by direct injection or aerosol exposure. The three groups of chickens showed no significant differences (p < 0.05) in mean death time after being directly challenged with NDV; however, in contrast to chickens in the non-transgenic and SPF groups, chickens in the transgenic group survived after aerosol exposure. Although the transgenic chickens did not survive after direct challenge, we found that the chickens expressing the 3D8 scFv survived aerosol exposure to NDV.

Conclusions

Our finding suggest that the 3D8 scFv could be a useful tool to prevent chickens from spreading NDV and control virus transmission.

SUBMITTER: Byun SJ

PROVIDER: S-EPMC7409462 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Publications

The 3D8 single chain variable fragment protein suppresses Newcastle disease virus transmission in transgenic chickens.

Byun Sung June SJ Choi Hoonsung H Sureshkumar Shanmugam S Yuk Seong-Su SS Kwon Jung-Hoon JH Noh Jin-Yong JY Jung Sun Keun SK Kim Jeom Sun JS Oh Keon Bong KB Yang Hyeon H Lee Gunsup G Lee Hwi-Cheul HC Woo Jae-Seok JS Song Chang-Seon CS

BMC veterinary research 20200806 1

<h4>Background</h4>The 3D8 single chain variable fragment (scFv) is a mini-antibody sequence that exhibits independent nuclease activity against all types of nucleic acids. In this research, crossing a 3D8 scFv G1 transgenic rooster with wild-type hens produced 3D8 scFv G<sub>2</sub> transgenic chickens to evaluate suppression of viral transmission.<h4>Result</h4>The transgenic chickens were identified using genomic PCR and immunohistochemistry. To evaluate Newcastle disease virus (NDV) protecti ...[more]

PMID: 32762754

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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

The 3D8 single chain variable fragment protein suppresses Newcastle disease virus transmission in transgenic chickens.

Background

Result

Conclusions

Publications

The 3D8 single chain variable fragment protein suppresses Newcastle disease virus transmission in transgenic chickens.

Similar Datasets

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