Expression and processing of the canine calicivirus capsid precursor

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 195-199 ◽  
Author(s):  
Yuichi Matsuura ◽  
Yukinobu Tohya ◽  
Mihoko Onuma ◽  
Frank Roerink ◽  
Masami Mochizuki ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Mammalian Cells ◽  
Expression System ◽  
Immunoblot Analysis ◽  
Site Directed Mutagenesis ◽  
Feline Calicivirus ◽  
Putative Cleavage Site ◽  
Mammalian Expression ◽  
Capsid Precursor ◽  
Infected Cells

The ORF2 product of canine calicivirus (CaCV) was identified and its processing in mammalian cells was analysed. Immunoblot analysis revealed the presence of the 75 kDa capsid precursor in addition to a 57 kDa capsid protein and a 22 kDa N-terminal polypeptide in CaCV-infected cells treated at an elevated temperature. When the CaCV ORF2 was expressed in a transient mammalian expression system, only the 75 kDa precursor was detected in immunoblot analysis, suggesting that no post-translational processing occurred in this system. However, the precursor was processed to a 57 kDa protein and a 22 kDa polypeptide by the proteinase of feline calicivirus (FCV) when this was co-expressed with ORF2. Processing was blocked by site-directed mutagenesis of the putative cleavage site in the capsid precursor. The results indicate that the proteinase of FCV can cleave the capsid precursor of CaCV to produce the mature capsid protein and that CaCV may have a similar proteinase.

scholarly journals Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3CproProteinase

1999 ◽  
Vol 73 (8) ◽  
pp. 6220-6227 ◽  
Author(s):  
Annette Martin ◽  
Danièle Bénichou ◽  
Shih-Fong Chao ◽  
Lisette M. Cohen ◽  
Stanley M. Lemon
Keyword(s):  
Capsid Protein ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
Recognition Sequence ◽  
C Terminus ◽  
Vaccinia Viruses ◽  
Capsid Protein Vp1 ◽  
Vp1 Capsid Protein

ABSTRACT Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the familyPicornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cproproteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cprorecognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase.

scholarly journals EKPRESI ANTIGEN Ag85B DARI Mycobacterium tuberculosis PADA GALUR SEL MAMALIA

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Sabar Pambudi ◽  
Tika Widayanti ◽  
Nadya Stephanie
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Recombinant Protein ◽  
Western Blot ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Culture Media ◽  
Mammalian Cell Culture ◽  
Expression System ◽  
Major Health Problem ◽  
Mammalian Expression

Expression of Mycobacterium tuberculosis Ag85B Antigen in Mammalian Cell CultureTuberculosis (TB) continues to be a major health problem worldwide, affecting millions of people each year. The only vaccine approved for the prevention of TB is Bacillus Calmette-Guérin (BCG). However, one of the limitations of BCG is that its preventive effect against pulmonary TB varies from person to person. Therefore, there arises a need for a new TB vaccine to replace BCG. This study aims to obtain the Ag85B recombinant protein which has characteristics similar to the native Ag85B antigen from Mycobacterium tuberculosis. In this study, we cloned and expressed recombinant Ag85B in mammalian cell culture. In the initial step, we cloned synthetic Ag85B into mammalian expression vector pFLAG-CMV4 and expressed the gene in CHO-K1 cells. Interestingly, a specific band around 30 kDa was observed in the culture media of transfected cells by Western blot analysis. The results from our research showed the potency of mammalian expression system to produce recombinant protein Ag85B for new TB vaccine candidate.Keywords: Ag85B, mammalian cells, tuberculosis, vaccine, expression ABSTRAKTuberkulosis (TB) terus menjadi salah satu masalah kesehatan dunia yang mempengaruhi jutaan manusia setiap tahun. Satu-satunya vaksin untuk TB yang ada adalah Bacillus Calmette-Guérin (BCG). Namun demikian, vaksin BCG ini memiliki kelemahan berupa terjadi efek preventif yang bervariasi dari satu individu terhadap individu lainnya.  Oleh sebab itu diperlukan pengembangan vaksin TB yang dapat menggantikan vaksin BCG yang sudah ada. Penelitian ini bertujuan memperoleh protein rekombinan Ag85B yang memiliki karakteristik mirip dengan antigen Ag85B native dari Mycobacterium tuberculosis. Pada penelitian ini, telah dilakukan kegiatan pengklonaan dan ekspresi gen Ag85B pada galur sel mamalia.  Pada tahap awal dilakukan pengklonaan gen sintesis Ag85B ke dalam plasmid pada sel mamalia pFLAG-CMV4 dan diekspresikan gennya pada sel CHO-K1. Hasil analisis Western blot menunjukan tersekresinya gen target berukuran 30 kDa pada media kultur dari sel mamalia yang ditransfeksi. Hasil dari penelitian ini menunjukkan potensi dari sistem ekpresi untuk protein rekombinan Ag85B pada galur sel mamalia sebagai kandidat vaksin TB yang baru.

scholarly journals Leader of the Capsid Protein in Feline Calicivirus Promotes Replication of Norwalk Virus in Cell Culture

2008 ◽  
Vol 82 (19) ◽  
pp. 9306-9317 ◽  
Author(s):  
Kyeong-Ok Chang ◽  
David W. George ◽  
John B. Patton ◽  
Kim Y. Green ◽  
Stanislav V. Sosnovtsev
Keyword(s):  
Cell Culture ◽  
Capsid Protein ◽  
Recombinant Plasmid ◽  
Fluorescent Protein ◽  
Feline Calicivirus ◽  
Norwalk Virus ◽  
Human Noroviruses ◽  
Infected Cells ◽  
Green Fluorescent ◽  
In Cells

ABSTRACT The inability to grow human noroviruses in cell culture has greatly impeded the studies of their pathogenesis and immunity. Vesiviruses, in the family Caliciviridae, grow efficiently in cell culture and encode a unique protein in the subgenomic region designated as leader of the capsid protein (LC). We hypothesized that LC might be associated with the efficient replication of vesiviruses in cell culture and promote the replication of human norovirus in cells. To test this hypothesis, a recombinant plasmid was engineered in which the LC region of feline calicivirus (FCV) was placed under the control of the cytomegalovirus promoter (pCI-LC) so that the LC protein could be provided in trans to replicating calicivirus genomes bearing a reporter gene. We constructed pNV-GFP, a recombinant plasmid containing a full-length NV genome with a green fluorescent protein (GFP) in the place of VP1. The transfection of pNV-GFP in MVA-T7-infected cells produced few GFP-positive cells detected by fluorescence microscopy and flow cytometry analysis. When pNV-GFP was cotransfected with pCI-LC in MVA-T7-infected cells, we observed an increase in the number of GFP-positive cells (ca. 3% of the whole-cell population). Using this cotransfection method with mutagenesis study, we identified potential cis-acting elements at the start of subgenomic RNA and the 3′ end of NV genome for the virus replication. We conclude that LC may be a viral factor which promotes the replication of NV in cells, which could provide a clue to growing the fastidious human noroviruses in cell culture.

In vitro expression of a mouse tissue specific glutathione-peroxidase-like protein lacking the selenocysteine can protect stably transfected mammalian cells against oxidative damage

1996 ◽  
Vol 74 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Patrick Vernet ◽  
Nicole Rigaudiére ◽  
Norbert Ghyselinck ◽  
Jean Pierre Dufaure ◽  
Joël R. Drevet
Keyword(s):  
Glutathione Peroxidase ◽  
Mammalian Cells ◽  
Expression System ◽  
Complete Sequence ◽  
Protein Domain ◽  
Mouse Tissue ◽  
Reading Frame ◽  
Mammalian Expression ◽  
Mouse Epididymis

The complete sequence of the mouse epididymal protein (MEP24) was cloned. It contains a 663 bp open-reading frame that, after conceptual translation, shows extensive identity with proteins belonging to the glutathione peroxidase (GPX) family. However, a major difference between GPX5 (MEP24) and other known GPXs concerns a protein domain known to be critical for GPX function. To find out what could be the physiological function of such a protein in the mouse epididymis, we have used a mammalian expression system to overexpress the GPX5 protein. Cells constitutively expressing the GPX5 protein were generated and assayed for their ability to metabolize regular substrates of GPX enzymes. Data presented here show that the GPX5-expressing cells can metabolize hydrogen peroxide in a manner that is consistent with a peroxidase activity. However, the substrate preference of the GPX5-expressing cells and their apparent insensitivity to a regular inhibitor of GPX enzymes suggest that the GPX5 protein belongs to a particular class of GPX proteins. Involvement of this protein in the physiology of the mouse epididymis is discussed.Key words: glutathione peroxidase (GPX), mouse epididymis, MEP24, GPX5 cDNA, selenocysteine, GPX5 polyclonal antibody, spermatozoa, CHO-KI cells.

scholarly journals Immunogenicity and Protective Activity of Pigeon Circovirus Recombinant Capsid Protein Virus-Like Particles (PiCV rCap-VLPs) in Pigeons (Columba livia) Experimentally Infected with PiCV

Vaccines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 98
Author(s):  
Huai-Ying Huang ◽  
Benji Brayan I. Silva ◽  
Shen-Pang Tsai ◽  
Ching-Yi Tsai ◽  
Yu-Chang Tyan ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Capsid Protein ◽  
Expression System ◽  
Genetic Material ◽  
Columba Livia ◽  
T Cell Proliferation ◽  
Virus Like Particles ◽  
Mammalian Expression ◽  
Wide Range

Pigeon circovirus (PiCV) is the most recurrent virus diagnosed in pigeons and is among the major causative agents of young pigeon disease syndrome (YPDS). Due to the lack of an established laboratory protocol for PiCV cultivation, development of prophylaxis is hampered. Alternatively, virus-like particles (VLPs), which closely resemble native viruses but lack the viral genetic material, can be generated using a wide range of expression systems and are shown to have strong immunogenicity. Therefore, the use of VLPs provides a promising prospect for vaccine development. In this study, transfected human embryonic kidney (HEK-293) cells, a mammalian expression system, were used to express the PiCV capsid protein (Cap), which is a major component of PiCV and believed to contain antibody epitopes, to obtain self-assembled VLPs. The VLPs were observed to have a spherical morphology with diameters ranging from 12 to 26 nm. Subcutaneous immunization of pigeons with 100 µg PiCV rCap-VLPs supplemented with water-in-oil-in-water (W/O/W) adjuvant induced specific antibodies against PiCV. Observations of the cytokine expression and T-cell proliferation levels in spleen samples showed significantly higher T-cell proliferation and IFN- γ expression in pigeons immunized with VLPs compared to the controls (p < 0.05). Experimentally infected pigeons that were vaccinated with VLPs also showed no detectable viral titer. The results of the current study demonstrated the potential use of PiCV rCap-VLPs as an effective vaccine candidate against PiCV.

scholarly journals A Comparative Investigation of the Bispecific Antibody: Expression in Expi293F Cells and E.coli

2021 ◽  
Vol 27 (3) ◽  
pp. 400-417
Author(s):  
Reza Moazzami ◽  
◽  
Hasan Mirzahosaini ◽  
Fatemeh Naddafi ◽  
Fatemeh Davami ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Expression System ◽  
Critical Issue ◽  
Comparative Investigation ◽  
Elisa Test ◽  
Industrial Scale ◽  
Binding Properties ◽  
Mammalian Expression ◽  
Study Results ◽  
Dual Character

Aims: Dual-Character antibodies can simultaneously target two surface markers. Blinatumomab is a C19 / CD3 antibody from the BiTE family (Bispecific T cell engager antibody) and was approved by the US Food and Drug Administration for clinical use. This antibody effectively targets malignant cells in patients with acute infoblastic leukemia. In the production of large quantities of such antibodies on an industrial scale, selecting the appropriate host remains a critical issue. Mammalian cells and strains of E.coli are the most common hosts for producing antibodies and antibody components on an industrial scale, respectively. Methods & Materials: In this study, a dual-specific antibody was used in the mammalian system of plasmid pcDNA3.1 (+) and for expression in the bacterial system of plasmid pET22b. The antibody produced in both systems was purified using nickel affinity resin under similar conditions. Next, SDS-PAGE and Western blot analysis was performed on both study samples. Finally, the binding properties of the antibody secreted from both systems were assessed by the ELISA test. Findings: The present study results suggested that antibodies produced by the mammalian expression system provided better binding properties than the expression system in bacteria. Conclusion: This study indicated that in the case of antibodies to two traits of the BiTE family, like Blinatombe, mammalian cells generate a more efficient and successful expression system; although the bacterium can produce much larger amounts of the antibody.

scholarly journals Heterologous expression and cell membrane localization of dinoflagellate rhodopsins in mammalian cells

2020 ◽  
Author(s):  
Minglei Ma ◽  
Xinguo Shi ◽  
Senjie Lin
Keyword(s):  
Cell Membrane ◽  
Proton Pump ◽  
Mammalian Cells ◽  
Expression System ◽  
Type I ◽  
Type Ii ◽  
Mammalian Expression ◽  
Domains Of Life ◽  
Targeting Signal ◽  
Large Groups

AbstractRhodopsins are now found in all domains of life, and are classified into two large groups: type II, found in animals and type I found in microbes including Bacteria, Archaea, and Eukarya. While type II rhodopsin functions in many photodependent signaling processes including vision, type I among others contains rhodopsins that function as a light-driven proton pump to convert light into ATP as in proteobacteria (named proteorhodopsin). Proteorhodopsin homologs have been documented in dinoflagellates, but their subcellular localizations and functions are still poorly understood. Even though sequence analyses suggest that it is a membrane protein, experimental evidence that dinoflagellate rhodopsins are localized on the plasma membrane or endomembranes is still lacking. As no robust dinoflagellate gene transformation tool is available, we used HEK 293T cells to construct a mammalian expression system for two dinoflagellate rhodopsin genes. The success of expressing these genes in the system shows that this mammalian cell type is suitable for expressing dinoflagellate genes. Immunofluorescence of the expressed protein locates these dinoflagellate rhodopsins on the cell membrane. This result indicates that the protein codons and membrane targeting signal of the dinoflagellate genes are compatible with the mammalian cells, and the proteins’ subcellular localization is consistent with proton pump rhodopsins.

scholarly journals Cleavage of the Feline Calicivirus Capsid Precursor Is Mediated by a Virus-Encoded Proteinase

1998 ◽  
Vol 72 (4) ◽  
pp. 3051-3059 ◽  
Author(s):  
Stanislav V. Sosnovtsev ◽  
Svetlana A. Sosnovtseva ◽  
Kim Y. Green
Keyword(s):  
Cleavage Site ◽  
Site Directed Mutagenesis ◽  
In Vitro Translation ◽  
Feline Calicivirus ◽  
Cdna Clones ◽  
Bacterial Cells ◽  
Structural Protein ◽  
Critical Determinant ◽  
Capsid Precursor

ABSTRACT Feline calicivirus (FCV), a member of theCaliciviridae, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3′-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates fortrans cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1′ position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.

Antigenic analysis of feline calicivirus capsid precursor protein and its deleted polypeptides produced in a mammalian cDNA expression system

1993 ◽  
Vol 30 (1) ◽  
pp. 17-26 ◽  
Author(s):  
Shin Yeon-Sil ◽  
Tohya Yukinobu ◽  
Oshikamo Ryoko ◽  
Kawaguchi Yasushi ◽  
Tomonaga Keizo ◽  
...  
Keyword(s):  
Expression System ◽  
Precursor Protein ◽  
Feline Calicivirus ◽  
Antigenic Analysis ◽  
Capsid Precursor ◽  
Cdna Expression

scholarly journals Snake deltavirus utilizes envelope proteins of different viruses to generate infectious particles

2019 ◽  
Author(s):  
Leonora Szirovicza ◽  
Udo Hetzel ◽  
Anja Kipar ◽  
Luis Martinez-Sobrido ◽  
Olli Vapalahti ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Particle Production ◽  
Helper Virus ◽  
Surface Proteins ◽  
Hepatitis D ◽  
Mammalian Expression ◽  
Viral Glycoproteins ◽  
Disease Associations ◽  
History Of ◽  
Infected Cells

ABSTRACTHepatitis D virus (HDV) is the only known human satellite virus, and it requires hepatitis B virus (HBV) to form infectious particles. Until 2018 HDV was the sole representative of the genus Deltavirus. The subsequent identification of HDV-like agents in non-human hosts indicated a much longer evolutionary history of deltaviruses than previously hypothesized. Interestingly, none of the HDV-like agents were found in co-infection with an HBV-like agent suggesting that these viruses use different helper virus(es). Here we show, using snake deltavirus (SDeV), that HBV represents only a single example of helper viruses for deltaviruses. We cloned the SDeV genome into a mammalian expression plasmid, and by transfection could initiate SDeV replication in cultured snake and mammalian cells. By superinfecting SDeV infected cells with arenaviruses, or by transfecting different viral surface proteins, we could induce infectious SDeV particle production. Our findings indicate that deltaviruses can likely use a multitude of helper viruses or even viral glycoproteins to form infectious particles. Persistent and recurrent infections would be beneficial for the spread of deltaviruses. It seems plausible that new human and veterinary disease associations with deltavirus infections will be identified in the future.

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