scholarly journals Recombinant scFv Antibodies against E Protein and N Protein of Severe Acute Respiratory Syndrome Virus

2004 ◽  
Vol 36 (8) ◽  
pp. 541-547 ◽  
Author(s):  
Hui Liu ◽  
Yan-Li Ding ◽  
Wei Han ◽  
Mei-Yun Liu ◽  
Rui-Yang Tian ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Binding Activity ◽  
Scfv Antibody ◽  
Respiratory Syndrome Virus ◽  
Single Chain ◽  
N Protein ◽  
Recombinant Scfv ◽  
E Protein ◽  
Equilibrium Dissociation ◽  
Site Binding

Abstract Three single chain antibodies (scFv) against the proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) were isolated by phage display from an scFv antibody library. Bio-panning was carried out against immobilized purified envelope (E) and nucleocapsid (N) proteins of SARS-CoV. Their binding activity and specificity to E or N protein of SARS-CoV were characterized by phage-ELISA. Two of them, B10 and C20, could recognize non-overlapping epitopes of the E protein according to the two-site binding test result. Clone A17 could recognize N protein. The sequence of the epitope or overlapping epitope of scFv antibody A17 was PTDSTDNNQNGGRNGARPKQRRPQ. The affinity (equilibrium dissociation constant, Kd) of SARS-CoV E protein was 5.7×10−8 M for B10 and 8.9×10−8 M for C20. The affinity of A17 for N protein was 2.1×10−6 M. All three scFv antibodies were purified with affinity chromatography and determined by Western blot.

scholarly journals Selection and characterization of scFv antibody against nucleocapsid protein of Porcine reproductive and respiratory syndrome virus

2020 ◽  
Vol 89 (1) ◽  
pp. 39-45
Author(s):  
Magdalena Krasna ◽  
Vladimír Celer
Keyword(s):  
Infectious Agent ◽  
Scfv Antibody ◽  
Respiratory Syndrome Virus ◽  
Single Chain ◽  
Single Chain Antibody ◽  
N Protein ◽  
E Coli ◽  
Metal Affinity ◽  
Binding Epitope

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widespread infectious agent in pigs. Nucleocapsid (N) protein of PRRSV has been identified as the most immunodominant viral protein. The main goal of the work was the selection and characterization of a single-chain antibody fragments (scFv) antibody specific to the N protein. Specific scFv antibody clone D5 was selected from the Tomlinson phagemid library and purified by immobilized metal affinity chromatography from the periplasmatic space of E. coli cells. The antibody was then characterized by sequencing and the ability to recognize the native virus N protein by Western blot and competitive ELISA. Pepscan analysis identified the position of the binding epitope between amino acids 62–84 of the N protein. Our study could help to improve the diagnostics and prevention of PRRSV in Central Europe.

scholarly journals Assembly of Severe Acute Respiratory Syndrome Coronavirus RNA Packaging Signal into Virus-Like Particles Is Nucleocapsid Dependent

2005 ◽  
Vol 79 (22) ◽  
pp. 13848-13855 ◽  
Author(s):  
Ping-Kun Hsieh ◽  
Shin C. Chang ◽  
Chu-Chun Huang ◽  
Ting-Ting Lee ◽  
Ching-Wen Hsiao ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Culture Medium ◽  
Binding Activity ◽  
Viral Rna ◽  
Structural Proteins ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Virus Like Particles ◽  
N Protein ◽  
Viral Genomic Rna

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm3. Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3′ noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.

scholarly journals Cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are non-essential for virus infectivity

2005 ◽  
Vol 86 (11) ◽  
pp. 3091-3096 ◽  
Author(s):  
Changhee Lee ◽  
Dongwan Yoo
Keyword(s):  
North American ◽  
Infectious Clone ◽  
Progeny Virus ◽  
Respiratory Syndrome Virus ◽  
Cysteine Residues ◽  
Virus Infectivity ◽  
Reading Frame ◽  
N Protein ◽  
Cytopathic Effects ◽  
E Protein

Porcine reproductive and respiratory syndrome virus (PRRSV) open reading frame (ORF) 2a contains a small internal ORF (2b) capable of encoding a protein of 73 aa, termed E protein. The function of E protein is currently unknown. The E protein possesses two cysteines at positions 49 and 54 that are highly conserved among North American isolates. In the present study, it was shown that E protein did not homodimerize with itself nor did it heterodimerize with the nucleocapsid (N) protein. However, E protein was interactive non-covalently with itself or with the N protein as shown by pull-down assays. The significance of the E protein cysteine residues on virus replication was determined using an infectious clone. Each cysteine was substituted by serine and the mutations were introduced into a full-length clone of PRRSV. When transfected into Marc-145 cells, all cysteine mutant clones induced PRRSV-specific cytopathic effects and produced infectious progeny virus. The data indicate that cysteine residues in the E protein are not essential for replication of North American genotype PRRSV.

scholarly journals Generation and Characterization of C305, a Murine Neutralizing scFv Antibody That Can Inhibit BLyS Binding to Its Receptor BCMA

2005 ◽  
Vol 37 (6) ◽  
pp. 415-420 ◽  
Author(s):  
Mei-Yun Liu ◽  
Wei Han ◽  
Yan-Li Ding ◽  
Tian-Hong Zhou ◽  
Rui-Yang Tian ◽  
...  
Keyword(s):  
B Lymphocyte ◽  
Neutralization Assay ◽  
Binding Activity ◽  
Scfv Antibody ◽  
Nucleotide Sequencing ◽  
Single Chain ◽  
Scfv Gene ◽  
Scfv Library ◽  
Soluble Scfv

Abstract B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pIII protein of M13 filamentous phage was screened using BLyS. After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4862-4869 ◽  
Author(s):  
Jörg F. Rippmann ◽  
Michaela Klein ◽  
Christian Hoischen ◽  
Bodo Brocks ◽  
Wolfgang J. Rettig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Binding Activity ◽  
Host Cells ◽  
Heterologous Proteins ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Active Product ◽  
Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

scholarly journals Serine 105 and 120 are important phosphorylation sites for porcine reproductive and respiratory syndrome virus N protein function

2018 ◽  
Vol 219 ◽  
pp. 128-135 ◽  
Author(s):  
Yao Chen ◽  
Xiulin Xing ◽  
Qi Li ◽  
Songlin Feng ◽  
Xiaoliang Han ◽  
...  
Keyword(s):  
Protein Function ◽  
Respiratory Syndrome Virus ◽  
Phosphorylation Sites ◽  
N Protein

scholarly journals Monoclonal Antibody-Based Antigen Capture Enzyme-Linked Immunosorbent Assay Reveals High Sensitivity of the Nucleocapsid Protein in Acute-Phase Sera of Severe Acute Respiratory Syndrome Patients

2005 ◽  
Vol 12 (1) ◽  
pp. 135-140 ◽  
Author(s):  
Biao Di ◽  
Wei Hao ◽  
Yang Gao ◽  
Ming Wang ◽  
Ya-di Wang ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Acute Phase ◽  
Detection Rate ◽  
Neutralization Test ◽  
Protein Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
N Protein ◽  
Antigen Capture ◽  
Healthy Donors

ABSTRACT Accurate and timely diagnosis of severe acute respiratory syndrome coronavirus (SARS-CoV) infection is a critical step in preventing another global outbreak. In this study, 829 serum specimens were collected from 643 patients initially reported to be infected with SARS-CoV. The sera were tested for the N protein of SARS-CoV by using an antigen capture enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies against the N protein of SARS-CoV and compared to 197 control serum samples from healthy donors and non-SARS febrile patients. The results of the N protein detection analysis were directly related to the serological analysis data. From 27 SARS patients who tested positive with the neutralization test, 100% of the 24 sera collected from 1 to 10 days after the onset of symptoms were positive for the N protein. N protein was not detected beyond day 11 in this group. The positive rates of N protein for sera collected at 1 to 5, 6 to 10, 11 to 15, and 16 to 20 days after the onset of symptoms for 414 samples from 298 serologically confirmed patients were 92.9, 69.8, 36.4, and 21.1%, respectively. For 294 sera from 248 serological test-negative patients, the rates were 25.6, 16.7, 9.3, and 0%, respectively. The N protein was not detected in 66 patients with cases of what was initially suspected to be SARS but serologically proven to be negative for SARS and in 197 serum samples from healthy donors and non-SARS febrile patients. The specificity of the assay was 100%. Furthermore, of 16 sera collected from four patients during the SARS recurrence in Guangzhou, 5 sera collected from 7 to 9 days after the onset of symptoms were positive for the N protein. N protein detection exhibited a high positive rate, 96 to 100%, between day 3 and day 5 after the onset of symptoms for 27 neutralization test-positive SARS patients and 298 serologically confirmed patients. The N protein detection rate continually decreased beginning with day 10, and N protein was not detected beyond day 19 after the onset of symptoms. In conclusion, an antigen capture ELISA reveals a high N protein detection rate in acute-phase sera of patients with SARS, which makes it useful for early diagnosis of SARS.

scholarly journals Rapid, sensitive, full genome sequencing of Severe Acute Respiratory Syndrome Virus Coronavirus 2 (SARS-CoV-2)

2020 ◽  
Author(s):  
Clinton R Paden ◽  
Ying Tao ◽  
Krista Queen ◽  
Jing Zhang ◽  
Yan Li ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Sanger Sequencing ◽  
Respiratory Syndrome Virus ◽  
Full Genome ◽  
Miseq Sequencing ◽  
Genomic Information ◽  
Rt Pcr ◽  
Full Genome Sequencing ◽  
High Quality ◽  
Global Pandemic

AbstractSARS-CoV-2 recently emerged, resulting a global pandemic. Rapid genomic information is critical to understanding transmission and pathogenesis. Here, we describe validated protocols for generating high-quality full-length genomes from primary samples. The first employs multiplex RT-PCR followed by MinION or MiSeq sequencing. The second uses singleplex, nested RT-PCR and Sanger sequencing.

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