scholarly journals A pH-Sensitive Heparin-Binding Sequence from Baculovirus gp64 Protein Is Important for Binding to Mammalian Cells but Not to Sf9 Insect Cells

2011 ◽  
Vol 86 (1) ◽  
pp. 484-491 ◽  
Author(s):  
C. Wu ◽  
S. Wang
Keyword(s):  
Mammalian Cells ◽  
Insect Cells ◽  
Ph Sensitive ◽  
Heparin Binding ◽  
Sf9 Insect Cells ◽  
Binding Sequence

Evaluation of the nucleopolyhedrovirus of Anticarsia gemmatalis as a vector for gene therapy in mammals

2020 ◽  
Vol 20 ◽  
Author(s):  
Cintia N. Parsza ◽  
Diego L. Mengual Gómez ◽  
Jorge Alejandro Simonin ◽  
Mariano Nicolás Belaich ◽  
Pablo Daniel Ghiringhelli
Keyword(s):  
Gene Therapy ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Autographa Californica ◽  
Anticarsia Gemmatalis ◽  
Agricultural Pests ◽  
Mammalian Cell Lines ◽  
Delivery Vector ◽  
Wide Range ◽  
Insect Pathogens

Background: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV) has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals, because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. Objective/Method: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. Results: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. Conclusion: Consequently, this insect pathogen is proposed as an alternative of non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

scholarly journals Expression and Immunogenicity of Two Recombinant Fusion Proteins Comprising Foot-and-Mouth Disease Virus Structural Protein VP1 and DC-SIGN-Binding Glycoproteins

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Xinsheng Liu ◽  
Jianliang Lv ◽  
Yuzhen Fang ◽  
Peng Zhou ◽  
Yanzhen Lu ◽  
...  
Keyword(s):  
Immune Responses ◽  
Disease Virus ◽  
Fusion Proteins ◽  
Insect Cells ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Vp1 Protein ◽  
Sf9 Insect Cells ◽  
Mouth Disease Virus ◽  
Foot And Mouth

Improving vaccine immunogenicity by targeting antigens to dendritic cells has recently emerged as a new design strategy in vaccine development. In this study, the VP1 gene of foot-and-mouth disease virus (FMDV) serotype A was fused with the gene encoding human immunodeficiency virus (HIV) membrane glycoprotein gp120 or C2-V3 domain of hepatitis C virus (HCV) envelope glycoprotein E2, both of which are DC-SIGN-binding glycoproteins. After codon optimization, the VP1 protein and the two recombinant VP1-gp120 and VP1-E2 fusion proteins were expressed in Sf9 insect cells using the insect cell-baculovirus expression system. Western blotting showed that the VP1 protein and two recombinant VP1-gp120 and VP1-E2 fusion proteins were correctly expressed in the Sf9 insect cells and had good reactogenicity. Guinea pigs were then immunized with the purified proteins, and the resulting humoral and cellular immune responses were analyzed. The VP1-gp120 and VP1-E2 fusion proteins induced significantly higher specific anti-FMDV antibody levels than the VP1 protein and stronger cell-mediated immune responses. This study provides a new perspective for the development of novel FMDV subunit vaccines.

scholarly journals Sesquiterpene Lactones from Artemisia absinthium. Biotransformation and Rearrangement of the Insect Antifeedant 3α-hydroxypelenolide

Plants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 891
Author(s):  
Braulio M. Fraga ◽  
Carmen E. Díaz ◽  
María Bailén ◽  
Azucena González-Coloma
Keyword(s):  
Cytotoxic Activity ◽  
Leptinotarsa Decemlineata ◽  
Spodoptera Littoralis ◽  
Culture Medium ◽  
Insect Cells ◽  
Sesquiterpene Lactones ◽  
Enzymatic System ◽  
Artemisia Absinthium ◽  
Sf9 Insect Cells ◽  
New Compounds

Three new compounds, the sesquiterpenes absilactone and hansonlactone and the acetophenone derivative ajenjol, have been isolated from a cultivated variety of Artemisia absinthium. In addition, the major lactone isolated, 3α-hydroxypelenolide, was biotransformed by the fungus Mucor plumbeus affording the corresponding 1β, 10α-epoxide. A cadinane derivative was formed by an acid rearrangement produced in the culture medium, but not by the enzymatic system of the fungus. Furthermore, 3α-hydroxypelenolide showed strong antifeedant effects against Leptinotarsa decemlineata and cytotoxic activity to Sf9 insect cells, while the biotransformed compounds showed antifeedant postingestive effects against Spodoptera littoralis.

scholarly journals Novel human-derived cell-penetrating peptides for specific subcellular delivery of therapeutic biomolecules

2005 ◽  
Vol 390 (2) ◽  
pp. 407-418 ◽  
Author(s):  
Catherine de Coupade ◽  
Antonio Fittipaldi ◽  
Vanessa Chagnas ◽  
Matthieu Michel ◽  
Sophie Carlier ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Mammalian Cells ◽  
Heparan Sulphate ◽  
Intracellular Delivery ◽  
Membrane Lipid ◽  
Cell Penetrating Peptides ◽  
Heparin Binding ◽  
Independent Manner ◽  
Cell Penetrating

Short peptide sequences that are able to transport molecules across the cell membrane have been developed as tools for intracellular delivery of therapeutic molecules. This work describes a novel family of cell-penetrating peptides named Vectocell® peptides [also termed DPVs (Diatos peptide vectors)]. These peptides, originating from human heparin binding proteins and/or anti-DNA antibodies, once conjugated to a therapeutic molecule, can deliver the molecule to either the cytoplasm or the nucleus of mammalian cells. Vectocell® peptides can drive intracellular delivery of molecules of varying molecular mass, including full-length active immunoglobulins, with efficiency often greater than that of the well-characterized cell-penetrating peptide Tat. The internalization of Vectocell® peptides has been demonstrated to occur in both adherent and suspension cell lines as well as in primary cells through an energy-dependent endocytosis process, involving cell-membrane lipid rafts. This endocytosis occurs after binding of the cell-penetrating peptides to extracellular heparan sulphate proteoglycans, except for one particular peptide (DPV1047) that partially originates from an anti-DNA antibody and is internalized in a caveolar independent manner. These new therapeutic tools are currently being developed for intracellular delivery of a number of active molecules and their potentiality for in vivo transduction investigated.

scholarly journals Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease

2001 ◽  
Vol 82 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Andres Merits ◽  
Lidia Vasiljeva ◽  
Tero Ahola ◽  
Leevi Kääriäinen ◽  
Petri Auvinen
Keyword(s):  
Mammalian Cells ◽  
Active Sites ◽  
Insect Cells ◽  
Semliki Forest Virus ◽  
Point Mutations ◽  
Proteolytic Processing ◽  
Wild Type ◽  
In Trans ◽  
Polyprotein Precursor

The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C478A, (P2CA) completely abolished the processing of P12CA34, P12CA3 and P2CA3. Co-expression of P23 and P12CA34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D6A, (P1234DA) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.

Purification of Recombinant p53 from Sf9 Insect Cells

p53 Protocols ◽  
2003 ◽  
pp. 17-28
Author(s):  
Xiu Zhu Sun ◽  
John Nguyen ◽  
Jamil Momand
Keyword(s):  
Insect Cells ◽  
Sf9 Insect Cells

scholarly journals A Newly Designed EGFP-2A Peptide Monocistronic Baculoviral Vector for Concatenating the Expression of Recombinant Proteins in Insect Cells

Processes ◽  
2019 ◽  
Vol 7 (5) ◽  
pp. 291 ◽  
Author(s):  
Chih-Yu Wu ◽  
Chao-Wei Huang ◽  
Yu-Shin Nai ◽  
Pei-Yu Chu ◽  
Chung-Hsiung Wang ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Fluorescent Protein ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
New Combination ◽  
Vector System ◽  
Baculovirus Expression Vector

Recombinant proteins produced by the baculovirus expression vector system (BVES) have been widely applied in the agricultural and medical fields. However, the procedure for protein expression is inefficient and needs to be improved. Herein, we propose a simple construct that incorporates a selectable marker (enhanced green fluorescent protein, EGFP) and a picorna viral-derived “self-cleaving” 2A-like peptide to separate the EGFP and target proteins in a monocistronic baculovirus vector to facilitate isolation of the recombinant baculovirus in the BVES. In this study, porcine adiponectin (ADN), a secreted, multimeric protein with insulin-sensitizing properties, was used to demonstrate its utility in our EGFP-2A-based expression system. EGFP and ADN were simultaneously expressed by a recombinant alphabaculovirus. Co-expression of EGFP facilitates the manipulation of the following processes, such as determining expression kinetics and harvesting ADN. The results showed that the 2A “self-cleaving” process does not interfere with EGFP activity or with signal peptide removal and the secretion of recombinant ADN. Posttranslational modifications, including glycosylation, of the recombinant ADN occurred in insect cells, and the formation of various multimers was further verified. Most importantly, the insect-produced ADN showed a similar bioactivity to that of mammalian cells. This concept provides a practical and economic approach that utilizes a new combination of alphabaculovirus/insect cell expression systems for future applications.

A COMPARISON OF THE BINDING OF ANTITHROMBIN III AND HEPARIN COFACTOR II TO HEPARINS, NATURALLY OCCURRING GLYCOSAMINOGLYCANS AND OTHER SULPHATED POLYMERS

1987 ◽  
Author(s):  
J Dawes ◽  
D S Pepper
Keyword(s):  
Molecular Weight ◽  
Chain Length ◽  
Antithrombin Iii ◽  
Molar Ratio ◽  
Heparin Binding ◽  
Dextran Sulphate ◽  
Heparin Cofactor Ii ◽  
Wide Range ◽  
Pentosan Polysulphate ◽  
Binding Sequence

Antithrombin III (ATIII) and heparin cofactor II (HCII) are currently thought to be the most important protein mediators of the anticoagulant and antithrombotic activities of glycosamino-glycans. A simple, quantitative method for assessing the affinity of a protein for a sulphated polymer in the liquid phase, based on competition with immobilised heparin, has been developed. Using this technique, the binding of ATIII and HCII to a wide range of glycosaminoglycans and other sulphated polymers have been compared, and the contributions to binding of size, degree of sulphation and backbone structure of the polymers analysed.In the presence of the high protein concentrations found in plasma, unfractionated heparin inhibited the binding of ATIII to immobilised heparin with a Ki of 1 x 10-6. Binding was destroyed by N-desulphation. 1 Results with a range of low molecular weight (LMW) heparins and heparan sulphates are consistent with the view that they all contain the ATIII-binding sequence, but at a lower molar ratio than heparin. Highly sulphated synthetic polymers such as dextran sulphate bound ATIII by a different mechanism, which was molecular weight-dependent.The affinity of HCII for heparins increased markedly with heparin chain length. Binding was largely, but not entirely, mediated by sulphate residues. HCII bound to heparan and dermatan sulphates with lower affinities than to heparin, and to synthetic sulphated polymers with similar or higher affinities. Pentosan polysulphate (SP54) bound HCII as effectively as did heparin. Binding of HCII to dextran sulphate was highly dependent on molecular weight. The affinity of HCII for a sulphated polymer appears to depend both on its chain length and density of sulphation.Thus the profiles of binding of ATIII and HCII to glycosaminoglycans and other sulphated polymers are quite different. This technique is useful both for investigating the interactions of existing therapeutic anticoagulants and assessing new products.

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