Preparation and characterization of a stable BHK-21 cell line constitutively expressing the Schmallenberg virus nucleocapsid protein

Zhang, Yongning; Wu, Shaoqiang; Song, Shanshan; Lv, Jizhou; Chen, Feng; Lin, Xiaoyan

doi:10.1016/j.mcp.2015.05.010

Cited by 7 publications

(11 citation statements)

References 28 publications

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“…Lentiviral vectors have been shown to be excellent tools for generating mammalian cell lines stably expressing exogenous genes (Liu et al 2016;Phanthanawiboon et al 2014;Zhang et al 2015). In the present study, we successfully generated a Vero cell line constitutively expressing the SBV-N protein using a lentivector-mediated gene transfer system in combination with puromycin selection.…”

Section: Discussionmentioning

confidence: 94%

“…Transduction of Vero cells with recombinant lentiviruses was performed as previously described (Zhang et al 2015). Briefly, Vero cells were seeded at a density of approximately 5 3 10 4 cells/well in a 24-well cell culture plate with DMEM containing 10% FBS.…”

Section: Lentiviral Transductionmentioning

confidence: 99%

“…Schmallenberg virus N fusion protein expressed in Vero-EGFP-SBV-N cells was purified under native conditions using Ni-NTA agarose according to the manufacturer's instructions (Qiagen 2003), with slight modifications (Zhang et al 2015). Briefly, approximately 4.2 9 10 8 cells were collected and lysed with RIPA lysis buffer.…”

Section: Protein Purificationmentioning

confidence: 99%

“…Immunofluorescence assay (IFA) Indirect IFA was performed on Vero cells using a previously described protocol (Zhang et al 2015). Briefly, Vero cells grown on coverslips were counterstained with Hoechst 33342 and then fixed with prechilled 100% ethanol for 15 min.…”

Section: Protein Purificationmentioning

confidence: 99%

“…In recent years, mammalian cells have become a powerful tool for the production of recombinant proteins, especially those needing post-translational modifications (Bandaranayake and Almo 2014;Büssow 2015;Kantardjieff and Zhou 2014). A variety of mammalian cell lines stably expressing exogenous genes were successfully generated using lentiviral vectors (Liu et al 2016;Phanthanawiboon et al 2014;Zhang et al 2015), which serve as efficient vehicles for transducing genes of interest into both dividing and nondividing cells, thereby achieving long-term stable expression of the transgenes (Nasri et al 2014).…”

Section: Introductionmentioning

confidence: 99%

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Generation and characterization of a potentially applicable Vero cell line constitutively expressing the Schmallenberg virus nucleocapsid protein

Zhang

Song

et al. 2017

Cytotechnology

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Schmallenberg virus (SBV) is a Culicoides-transmitted orthobunyavirus that poses a threat to susceptible livestock species such as cattle, sheep and goats. The nucleocapsid (N) protein of SBV is an ideal diagnostic antigen for the detection of viral infection. In this study, a stable Vero cell line, Vero-EGFP-SBV-N, constitutively expressing the SBV-N protein was established using a lentivirus system combined with puromycin selection. This cell line spontaneously emitted green fluorescent signals distributed throughout the cytoplasm, in which the expression of SBV-N fusion protein was confirmed by western blot analysis. The expression of SBV-N protein in Vero-EGFP-SBV-N cells was stable for more than fifty passages without puromycin pressure. The SBV-N fusion protein contained both an N-terminal enhanced green fluorescent protein (EGFP) tag and a C-terminal hexa-histidine (6 × His) tag, by which the N protein was successfully purified using Ni-NTA affinity chromatography. The cell line was further demonstrated to be reactive with SBV antisera and an anti-SBV monoclonal antibody in indirect immunofluorescence assays. Taken together, our results demonstrate that the Vero-EGFP-SBV-N cell line has potential for application in the serological diagnosis of SBV infection.

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Section: Discussionmentioning

confidence: 94%

Section: Lentiviral Transductionmentioning

confidence: 99%

Section: Protein Purificationmentioning

confidence: 99%

Section: Protein Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Generation and characterization of a potentially applicable Vero cell line constitutively expressing the Schmallenberg virus nucleocapsid protein

Zhang

Song

et al. 2017

Cytotechnology

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Development and validation of a real-time RT-PCR assay for the quantification of rabies virus as quality control of inactivated rabies vaccines

Moreira

Pereira

Gimenez

et al. 2019

Journal of Virological Methods

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Characterization and Double-Antibody Sandwich ELISA Application of a Monoclonal Antibody Against Akabane Virus Nucleocapsid Protein

Chen

Wang

Wei

et al. 2023

Journal of AOAC International

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Background Akabane virus (AKAV) is a Culicoides-borne Orthobunyavirus that is teratogenic to the fetus of cattle and small ruminant species. Objectives This study aimed to develop an effective diagnostic assay for the detection of AKAV using produced monoclonal antibody (mAb). Methods Firstly, the mAb against N protein of AKAV was produced and characterized by western blot (WB) and indirect immunofluorescence (IFA) assays. Then, the linear epitope of AKAV N protein against the mAb was identified and the mAb was applied to establish a double antibody sandwich ELISA (DAS-ELISA). Results One AKAV N-reactive monoclonal mAb was generated and designated as 2D3. WB and IFA assays indicated that 2D3 could react with both recombinant N protein and AKAV isolate TJ2016. The linear epitope recognized by mAb 2D3 was located at amino acids 168–182 of AKAV N protein. The double antibody sandwich ELISA (DAS-ELISA) established based mAb 2D3 was able to detect both the purified AKAV N protein (with detection limit of 6.25 ng/ml) and AKAV-infected cell culture supernatant (with detection limit of 250 TCID50/ml). Conclusion Taken together, we successfully prepared a mAb 2D3 against AKAV N protein and identified its corresponding linear epitope, and then established a DAS-ELISA for the detection of AKAV antigen. Highlights A produced mAb against AKAV N protein was used to define a linear epitope of AKAV and establish a DAS-ELISA for AKAV antigen detection.

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Preparation and characterization of a stable BHK-21 cell line constitutively expressing the Schmallenberg virus nucleocapsid protein

Cited by 7 publications

References 28 publications

Generation and characterization of a potentially applicable Vero cell line constitutively expressing the Schmallenberg virus nucleocapsid protein

Generation and characterization of a potentially applicable Vero cell line constitutively expressing the Schmallenberg virus nucleocapsid protein

Development and validation of a real-time RT-PCR assay for the quantification of rabies virus as quality control of inactivated rabies vaccines

Characterization and Double-Antibody Sandwich ELISA Application of a Monoclonal Antibody Against Akabane Virus Nucleocapsid Protein

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