scholarly journals Construction of an Enzymatically-Conjugated DNA Aptamer–Protein Hybrid Molecule for Use as a BRET-Based Biosensor

2020 ◽  
Vol 10 (21) ◽  
pp. 7646
Author(s):  
Masayasu Mie ◽  
Rena Hirashima ◽  
Yasumasa Mashimo ◽  
Eiry Kobatake
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Resonance Energy ◽  
Porcine Circovirus Type ◽  
Replication Initiation ◽  
Dna Aptamer ◽  
Porcine Circovirus ◽  
Dna Aptamers ◽  
Hybrid Molecule

DNA-protein conjugates are useful molecules for construction of biosensors. Herein, we report the development of an enzymatically-conjugated DNA aptamer–protein hybrid molecule for use as a bioluminescence resonance energy transfer (BRET)-based biosensor. DNA aptamers were enzymatically conjugated to a fusion protein via the catalytic domain of porcine circovirus type 2 replication initiation protein (PCV2 Rep) comprising residues 14–109 (tpRep), which was truncated from the full catalytic domain of PCV2 Rep comprising residues 1–116 by removing the flexible regions at the N- and C-terminals. For development of a BRET-based biosensor, we constructed a fusion protein in which tpRep was positioned between NanoLuc luciferase and a fluorescent protein and conjugated to single-stranded DNA aptamers that specifically bind to either thrombin or lysozyme. We demonstrated that the BRET ratios depended on the concentration of the target molecules.

scholarly journals Enhancement of correct protein folding in vivo by a non-lytic baculovirus

2004 ◽  
Vol 382 (2) ◽  
pp. 695-702 ◽  
Author(s):  
Yu HO ◽  
Huei-Ru LO ◽  
Tzu-Ching LEE ◽  
Carol P. Y. WU ◽  
Yu-Chan CHAO
Keyword(s):  
Energy Transfer ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Random Mutagenesis ◽  
Resonance Energy ◽  
Vector System ◽  
Enhanced Yellow Fluorescent Protein

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP–LUC–ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

scholarly journals Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein Promotes Protective Immune Responses in Mice

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0129617 ◽  
Author(s):  
Chunyan Zhang ◽  
Shanshan Zhu ◽  
Li Wei ◽  
Xu Yan ◽  
Jing Wang ◽  
...  
Keyword(s):  
Immune Responses ◽  
Fusion Protein ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus

scholarly journals Evaluation of a Virus-like Nanoparticle Porcine Circovirus Type-2 (PCV2) Capsid Protein Fused with the Pig Immunoglobulin Fc Fragment as a Novel Vaccine Candidate against PCV2 in Mice

Vaccines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1128
Author(s):  
Qingping Luo ◽  
Waqas Ahmed ◽  
Yichen Dai ◽  
Ali Mohsin ◽  
Haifeng Hang ◽  
...  
Keyword(s):  
Immune Responses ◽  
Fusion Protein ◽  
Capsid Protein ◽  
Ex Vivo ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus ◽  
Cell Reactivity ◽  
Fc Fragment

Porcine circovirus Type 2 (PCV2) is a primary etiological pathogen of post-weaning multi-systemic wasting syndrome (PMWS). The capsid protein of PCV2 is the crucial immunogenic protein which can induce antibody generation and immune responses. However, there is still a lack of efficient PCV2 vaccines with high immunogenicity. In the current study, we developed a novel engineered PCV2 capsid (∆1-41aa)-pFc fusion protein (PCFP), which comprised a truncated capsid protein of PCV2 and a porcine IgG Fc fragment, fused to the capsid protein of PCV2 at the C-terminus. We found that this novel fusion protein could auto-assemble into virus-like nanoparticles with an estimated mean diameter of 22.6 nm, characterized by transmission electron microscopy. Immunization of BALB/c mice with this fusion protein significantly increased the production levels of anti-PCV2-capsid protein antibody in serum. Besides, the virus-like nanoparticles, PCFP was demonstrated to induce efficient cellular immune responses in mice, as evident by the high specific T cell reactivity to the PCFP fusion protein and the high production of the immune cytokines IFN-γ and IL-10 in an ex vivo re-stimulation system. Collectively, these findings demonstrate that the PCV2 truncated capsid subunit Fc-fusion protein can induce both cellular and humoral immune responses, and it displays great application potential.

scholarly journals Immunity Elicited by an Experimental Vaccine Based on Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein in Piglets

PLoS ONE ◽  
2016 ◽  
Vol 11 (2) ◽  
pp. e0147432 ◽  
Author(s):  
Shanshan Zhu ◽  
Chunyan Zhang ◽  
Jing Wang ◽  
Li Wei ◽  
Rong Quan ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus

scholarly journals Cytotoxicity of ORF3 Proteins from a Nonpathogenic and a Pathogenic Porcine Circovirus

2010 ◽  
Vol 84 (21) ◽  
pp. 11440-11447 ◽  
Author(s):  
Mark Chaiyakul ◽  
Karolynn Hsu ◽  
Rkia Dardari ◽  
Frank Marshall ◽  
Markus Czub
Keyword(s):  
Cell Death ◽  
Fluorescent Protein ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Cell Types ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus ◽  
Parp Cleavage ◽  
Orf3 Protein ◽  
Induced Apoptosis

ABSTRACT Porcine circovirus type 2 (PCV2) infection is associated with significant and serious swine diseases worldwide, while PCV1 appears to be a nonpathogenic virus. Previous studies demonstrated that the ORF3 protein of PCV2 (PCV2ORF3) was involved in PCV2 pathogenesis via its proapoptotic capability (J. Liu, I. Chen, Q. Du, H. Chua, and J. Kwang, J. Virol. 80:5065-5073, 2006). If PCV2ORF3-induced apoptosis is a determinant of virulence, PCV1ORF3 is hypothesized to lack this ability. The properties of PCV1 and PCV2 ORF3, expressed as fusion proteins to an enhanced green fluorescent protein (eGFP), were characterized with regard to their ability to cause cellular morphological changes, detachment, death, and apoptosis. PCV1ORF3 significantly induced more apoptotic cell death and was toxic to more different cell types than PCV2ORF3 was. PCV1ORF3-associated cell death was caspase dependent. PCV1ORF3 also induced poly(ADP-ribose) polymerase 1 (PARP) cleavage; however, whether PARP was involved in cell death requires further studies. Truncation of PCV1 and elongation of PCV2 ORF3 proteins revealed that the first 104 amino acids contain a domain capable of inducing cell death, whereas the C terminus of PCV1ORF3 contains a domain possibly responsible for enhancing cell death. These results suggest that the pathogenicity of PCV2 for pigs is either not determined or not solely determined by the ORF3 protein.

scholarly journals Correction to: A nanobody-horseradish peroxidase fusion protein-based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yang Mu ◽  
Cunyu Jia ◽  
Xu Zheng ◽  
Haipeng Zhu ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Horseradish Peroxidase ◽  
Fusion Protein ◽  
Rapid Detection ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Competitive Elisa ◽  
Porcine Circovirus

An amendment to this paper has been published and can be accessed via the original article.

scholarly journals A nanobody‐horseradish peroxidase fusion protein‐based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yang Mu ◽  
Cunyu Jia ◽  
Xu Zheng ◽  
Haipeng Zhu ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Horseradish Peroxidase ◽  
Fusion Protein ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Competitive Elisa ◽  
Porcine Circovirus ◽  
Serum Samples ◽  
Porcine Serum ◽  
Commercial Kits

Abstract Background The widespread popularity of porcine circovirus type 2(PCV2) has seriously affected the healthy development of the pig industry and caused huge economic losses worldwide. A rapid and reliable method is required for epidemiological investigation and evaluating the effect of immunization. However, the current methods for PCV2 antibody detection are time-consuming or very expensive and rarely meet the requirements for clinical application. we have constructed the platform for expressing the nanobody(Nb)‑horseradish peroxidase(HRP) fusion protein as an ultrasensitive probe to detect antibodies against the Newcastle disease virus(NDV), previously. In the present work, an Nb-HRP fusion protein-based competitive ELISA(cELISA) for rapid and simple detection antibodies against PCV2 was developed using this platform to detect anti-PCV2 antibodies in clinical porcine serum. Results Using phage display technology, 19 anti-PCV2-Cap protein nanobodies were screened from a PCV2-Cap protein immunized Bactrian camel. With the platform, the PCV2-Nb15‑HRP fusion protein was then produced and used as a sensitive reagent for developing a cELISA to detect anti‑PCV2 antibodies. The cut‑off value of the cELISA is 20.72 %. Three hundreds and sixty porcine serum samples were tested by both newly developed cELISA and commercial kits. The sensitivity and specificity were 99.68 % and 95.92 %, respectively. The coincidence rate of the two methods was 99.17 %. When detecting 620 clinical porcine serum samples, a good consistent (kappa value = 0.954) was found between the results of the cELISA and those of commercial kits. Conclusions In brief, the newly developed cELISA based PCV2-Nb15‑HRP fusion protein is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PCV2 vaccine efficacy and the indirect diagnosis of PCV2 infection.

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