scholarly journals Impact of Molecular Modification on the Efficiency of Recombinant Baculovirus Vector Invasion to Mammalian Cells and Its Immunogenicity in Mice

Viruses ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 140
Author(s):  
Hao Zheng ◽  
Yong Pan ◽  
Xiong Wang ◽  
Weibin Tian ◽  
Lunguang Yao ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
High Efficiency ◽  
Fluorescent Proteins ◽  
Surface Display ◽  
Recombinant Baculovirus ◽  
Fold Increase ◽  
Host Cells ◽  
Baculovirus Vector ◽  
Display Technology ◽  
Expression Activity

The baculovirus display system (BDS), an excellent eukaryotic surface display technology that offers the advantages of safety, efficiency, and economy, is widely used in biomedicine. A previous study using rBacmid-Δgp64-ires-gp64 expressed in low copy numbers of the gp64 gene achieved high-efficiency expression and co-display of three fluorescent proteins (GFP, YFP, and mCherry). However, low expression of GP64 in recombinant baculoviruses also reduces the efficiency of recombinant baculovirus transduction into mammalian cells. In addition, the baculovirus promoter has no expression activity in mammalian cells and thus cannot meet the application requirements of baculoviral vectors for the BDS. Based on previous research, this study first determined the expression activity of promoters in insect Spodoptera frugiperda 9 cells and mammalian cells and successfully screened the very early promoter pie1 to mediate the co-expression of multiple genes. Second, utilizing the envelope display effect of the INVASIN and VSVG proteins, the efficiency of transduction of recombinant baculovirus particles into non-host cells was significantly improved. Finally, based on the above improvement, a recombinant baculovirus vector displaying four antigen proteins with high efficiency was constructed. Compared with traditional BDSs, the rBacmid-Δgp64 system exhibited increased display efficiency of the target protein by approximately 3-fold and induced an approximately 4-fold increase in the titer of serum antibodies to target antigens in Bal B/c mice. This study systematically explored the application of a new multi-gene co-display technology applicable to multi-vaccine research, and the results provide a foundation for the development of novel BDS technologies.

scholarly journals Surface Display Technology for Biosensor Applications: A Review

Sensors ◽  
2020 ◽  
Vol 20 (10) ◽  
pp. 2775 ◽  
Author(s):  
Min Park
Keyword(s):  
Mammalian Cells ◽  
Surface Display ◽  
Cell Surface Display ◽  
Bacterial Surface ◽  
Yeast Cell Surface ◽  
Bacterial Surface Display ◽  
Display Technology ◽  
Almost All ◽  
Display Systems ◽  
Biosensor Applications

Surface display is a recombinant technology that expresses target proteins on cell membranes and can be applied to almost all types of biological entities from viruses to mammalian cells. This technique has been used for various biotechnical and biomedical applications such as drug screening, biocatalysts, library screening, quantitative assays, and biosensors. In this review, the use of surface display technology in biosensor applications is discussed. In detail, phage display, bacterial surface display of Gram-negative and Gram-positive bacteria, and eukaryotic yeast cell surface display systems are presented. The review describes the advantages of surface display systems for biosensor applications and summarizes the applications of surface displays to biosensors.

scholarly journals Identification of OmpU of Vibrio vulnificus as a Fibronectin-Binding Protein and Its Role in Bacterial Pathogenesis

2006 ◽  
Vol 74 (10) ◽  
pp. 5586-5594 ◽  
Author(s):  
Sung Young Goo ◽  
Hyun-Ju Lee ◽  
Woo Hwang Kim ◽  
Kyu-Lee Han ◽  
Dae-Kyun Park ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Vibrio Vulnificus ◽  
Outer Membrane Proteins ◽  
Lethal Dose ◽  
Fold Increase ◽  
Host Cells ◽  
Wild Type ◽  
Binding Assays ◽  
Exogenous Addition ◽  
Main Components

ABSTRACT Vibrio vulnificus is a pathogenic bacterium that causes gastroenteritis and primary septicemia. To identify factors involved in microbial adherence to the host cells, we investigated bacterial proteins capable of binding to fibronectin, one of the main components comprised of the extracellular matrix of mammalian cells. A protein of ∼35 kDa was purified from the extracts of V. vulnificus by its property to bind to immobilized fibronectin. This protein was identified as OmpU, one of the major outer membrane proteins of V. vulnificus. In binding assays using immobilized fibronectin, the number of ompU mutant cells bound to fibronectin was only 4% of that of wild-type cells bound to fibronectin. In addition, the exogenous addition of antibodies against OmpU resulted in a decreased ability of wild-type V. vulnificus to adhere to fibronectin. The ompU mutant was also defective in its adherence to RGD tripeptide (5% of the adherence of the wild type to RGD), cytoadherence to HEp-2 cells (7% of the adherence of the wild type to HEp-2), cytotoxicity to cell cultures (39% of the cytotoxicity of the wild type), and mortality in mice (10-fold increase in the 50% lethal dose). The ompU mutant complemented with the intact ompU gene restored its abilities for adherence to fibronectin, RGD tripeptide, and HEp-2 cells; cytotoxicity to HEp-2 cells; and mouse lethality. This study indicates that OmpU is an important virulence factor involved in the adherence of V. vulnificus to the host cells.

scholarly journals High-efficiency retron-mediated single-stranded DNA production in plants

2021 ◽  
Author(s):  
Wenjun Jiang ◽  
Gundra Sivakrishna Rao ◽  
Rashid Aman ◽  
Haroon Butt ◽  
Radwa Kamel ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genome Engineering ◽  
High Efficiency ◽  
Plant Biotechnology ◽  
Fold Increase ◽  
Gene Encoding ◽  
Single Stranded Dna ◽  
Homology Directed Repair ◽  
Potential Applications

ABSTRACT Background: Retrons are a class of retroelements that produce multicopy single-stranded DNA (msDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the over-production of single-stranded DNA (ssDNA), genome engineering, and directed evolution in bacteria, yeast, and mammalian cells. However, no studies have shown retron-mediated ssDNA production in plants, which could unlock potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Main methods and major results: Here, we demonstrated retron-mediated over-production of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated CRISPR-retron-coupled ssDNA over-production and targeted HDR in N. benthamiana. Conclusion: We present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications.

scholarly journals Error-prone mutagenesis detected in mammalian cells by a shuttle vector containing the supF gene of Escherichia coli.

1984 ◽  
Vol 4 (10) ◽  
pp. 2227-2230 ◽  
Author(s):  
S Sarkar ◽  
U B Dasgupta ◽  
W C Summers
Keyword(s):  
Escherichia Coli ◽  
Kidney Cell ◽  
Mammalian Cells ◽  
Mutation Frequency ◽  
Shuttle Vector ◽  
Fold Increase ◽  
Host Cells ◽  
Kidney Cell Line ◽  
Monkey Kidney Cell ◽  
Supf Gene

When a shuttle vector containing a tyrosine suppressor tRNA (supF) gene as a target for mutagenesis replicated in a monkey kidney cell line, the frequency of SupF+ mutations was 2.3 +/- 0.5 x 10(-3). When the host cells were treated with ethyl methanesulfonate 40 h before transfection, a 10-fold increase in SupF+ mutation frequency was observed. These results supported the hypothesis that a damage-inducible mutagenic pathway exists in mammalian cells and also demonstrated the utility of this shuttle vector for the study of mutagenesis in mammalian cells.

Error-prone mutagenesis detected in mammalian cells by a shuttle vector containing the supF gene of Escherichia coli

1984 ◽  
Vol 4 (10) ◽  
pp. 2227-2230
Author(s):  
S Sarkar ◽  
U B Dasgupta ◽  
W C Summers
Keyword(s):  
Escherichia Coli ◽  
Kidney Cell ◽  
Mammalian Cells ◽  
Mutation Frequency ◽  
Shuttle Vector ◽  
Fold Increase ◽  
Host Cells ◽  
Kidney Cell Line ◽  
Monkey Kidney Cell ◽  
Supf Gene

When a shuttle vector containing a tyrosine suppressor tRNA (supF) gene as a target for mutagenesis replicated in a monkey kidney cell line, the frequency of SupF+ mutations was 2.3 +/- 0.5 x 10(-3). When the host cells were treated with ethyl methanesulfonate 40 h before transfection, a 10-fold increase in SupF+ mutation frequency was observed. These results supported the hypothesis that a damage-inducible mutagenic pathway exists in mammalian cells and also demonstrated the utility of this shuttle vector for the study of mutagenesis in mammalian cells.

scholarly journals Feasibility of using a dual-promoter recombinant baculovirus vector to coexpress EGFP and GDNF in mammalian cells

2014 ◽  
Vol 7 (6) ◽  
pp. 1549-1554
Author(s):  
JIANZHANG WANG ◽  
JUN WANG ◽  
CHANGPING CAI ◽  
SHILI WANG ◽  
SHUAI LIU ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Recombinant Baculovirus ◽  
Baculovirus Vector ◽  
Dual Promoter

scholarly journals Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

2020 ◽  
Author(s):  
Srijit Mukherjee ◽  
Sheng-Ting Hung ◽  
Nancy Douglas ◽  
Premashis Manna ◽  
Connor Thomas ◽  
...  
Keyword(s):  
Rate Constant ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Point Mutations ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Radiative Rate ◽  
Low Cytotoxicity ◽  
Molecular Brightness

AbstractThe development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and appropriate localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, FusionRed-MQV, which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (~140,000 M−1cm−1), increased radiative rate constant and reduced non-radiative rate constant with respect to its precursor. The properties of FusionRed-MQV derive from three mutations - M42Q, C159V and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the phenol and the acylimine ends of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the phenol end of the chromophore were mutated. The M42Q mutation is located near the acylimine end of the chromophore and was discovered using site-directed mutagenesis guided by x-ray crystal structures. FusionRed-MQV exhibits 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells (based on FACS) compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells.

scholarly journals Baculovirus-Mediated Gene Delivery into Mammalian Cells Does Not Alter Their Transcriptional and Differentiating Potential but Is Accompanied by Early Viral Gene Expression

2006 ◽  
Vol 80 (8) ◽  
pp. 4135-4146 ◽  
Author(s):  
Christos Kenoutis ◽  
Rodica C. Efrose ◽  
Luc Swevers ◽  
Alexandros A. Lavdas ◽  
Maria Gaitanou ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Schwann Cells ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Primary Cultures ◽  
Recombinant Baculovirus ◽  
Hek293 Cells ◽  
Viral Gene ◽  
Transcriptional Responses ◽  
Baculovirus Infection

ABSTRACT Gene delivery to neural cells is central to the development of transplantation therapies for neurological diseases. In this study, we used a baculovirus derived from the domesticated silk moth, Bombyx mori, as vector for transducing a human cell line (HEK293) and primary cultures of rat Schwann cells. Under optimal conditions of infection with a recombinant baculovirus containing the reporter green fluorescent protein gene under mammalian promoter control, the infected cells express the transgene with high efficiency. Toxicity assays and transcriptome analyses suggest that baculovirus infection is not cytotoxic and does not induce differential transcriptional responses in HEK293 cells. Infected Schwann cells retain their characteristic morphological and molecular phenotype as determined by immunocytochemistry for the marker proteins S-100, glial fibrillary acidic protein, and p75 nerve growth factor receptor. Moreover, baculovirus-infected Schwann cells are capable of differentiating in vitro and express the P0 myelination marker. However, transcripts for several immediate-early viral genes also accumulate in readily detectable levels in the transduced cells. This transcriptional activity raises concerns regarding the long-term safety of baculovirus vectors for gene therapy applications. Potential approaches for overcoming the identified problem are discussed.

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