• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.355-362
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• 2000

The Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) gene in the pHLA-21 plasmid was inserted into a baculovirus (Hyphantria cunea nuclear polyhedrosis virus) expression vector (lacZ-HcNPV) to construct a recombinant virus gB-HcNPV expressing gB. Spodoptera frugiperda cells infected with this recombinant virus synthesized and processed gB of approximately 120 kDa, which cross-reacted with the monoclonal antibody to gB. The recombinant gB was identified on the membrane of the insect cells using an immunofluorescence assay. Antibodies to this recombinant raised in mice recognize the viral gB and neutralized the infectivity of the HSV-1 in vitro. These results show that the gB gene has the potential to be expressed in insect cells. They also demonstrate that it is possible to produce a mature protein by gene transfer in eukaryotic cells, and indicate the utility of the lacZ-HcNPV-insect cell system for producing and characterizing eukaryotic proteins. Furthermore, the neutralizing antibodies would appear to protect mice against HSV. Accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.

• Biomedical Science Letters
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• v.15 no.1
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• pp.55-60
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• 2009

Insect cells such as Spodoptera frugiperda Clone 9 (Sf9) cells are widely chosen as the host for heterologous expression of a mammalian sugar transport protein using the baculovirus expression system. Characterization of the expressed protein is expected to include assay of its function, including its ability to transport sugars and to bind inhibitory ligands such as cytochalasin B. It is therefore very important first to establish the transport characteristics and other properties of the endogenous sugar transport proteins of the host insect cells. However, very little is known of the transport characteristics of Sf9 cells, although their ability to grow on TC-100 medium strongly suggested the presence of endogenous glucose transport system. In order to investigate the substrate and inhibitor recognition properties of the Sf9 cell transporter, the ability of pentoses to inhibit 2-deoxy-D-glucose (2dGlc) transport was investigated by measuring inhibition constants $(K_i)$. To determine the time period over which of sugar into the Sf cells was linear, the uptake of 2dGlc 0.1mM extracellular concentration was measured over periods ranging from 30 seconds to 30 minutes. The uptake was linear for at least 2 minutes at the concentration, implying that uptake made over a 1 minute time course would reflect initial rates of the sugar uptake. The data have also revealed the existence of a saturable transport system for pentose uptake by the insect cells. The transport was inhibited by D-xylose and D-ribose, although not as effective as hexoses. However, L-xylose had a little effect on 2dGlc transport in the Sf9 cells, indicating that the transport is stereoselective. Unlike the human erythrocyte-type glucose transport system, D-ribose had a somewhat greater apparent affinity for the Sf9 cell transporter than D-xylose. It is therefore concluded that Sf9 cells contain an endogenous sugar transport activity that in some aspects resembled the human erythrocyte-type counterpart, although the Sf9 and human transport systems do differ in their affinity for cytochalasin B.

• Biomedical Science Letters
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• v.15 no.1
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• pp.37-45
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• 2009

Insect cell cultures have become important tools in the production of biological substances for use in a variety of research, human and veterinary medicine, and pest control applications. These applications often require the introduction of foreign DNA into the cells and have generally used methods originally developed for use with human and other mammalian cell cultures. While these methods can be successfully employed, they are often less efficient with insect cells and frequently involve complex procedures or require specialized equipment. Even when they do work, they may require substantial modification because of differences in the culture medium or growth patterns of insect cells. In this study, We have optimized transfection conditions of Sf9 cell line using insect expression vector pIZT/V5-His which expresses green fluorescent protein effectively. Human stem cell factor (hSCF) is a glycoprotein that plays a key role in hematopoiesis acting both as a positive and negative regulator, often in synergy with other cytokines. It also plays a key role in mast cell development, gametogenesis, and melanogenesis. It can exist in membrane-bound form and in proteolytically released soluble form. As determined by an enzyme-linked immunosorbent assay performed, hSCF level in supernatant averaged 995ng/ml. The human hSCF was partially purified by immunoaffinity chromatography and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The results show that the hSCF has N-linked carbohydrate and corresponds to the soluble form, at or about 223 amino acids in length. The findings suggest functional importance for soluble hSCF in cells.

• YAKHAK HOEJI
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• v.47 no.6
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• pp.410-416
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• 2003

Autotaxin(ATX) was originally purified from conditioned media of A2058 human melanoma cells and shown to be a potent cell motility-stimulating factor, possessing a type II nucleotide pyrophosphatase/phosphodiesterase (NPP2) activity. Recombinant ATX has recently demonstrated that human plasma lysophosholipase D is identical to ATX and uses lysophosphatidylcholine as a substrate to mediate various biological functions including tumor cell growth and motility through G-protein coupled receptor. However, despite pivotal roles of ATX on physiological or pathophysiological states, the production of ATX is solely depends on complicated purification method which employs multiple column steps, but resulted in very poor yield. This limited the use of ATX for extensive analysis. We, therefore, expressed six histidine-tagged recombinant human ATX(His-ATX) in High Five TM insect cells to improve the generation of ATX and to make simple the purification of ATX. The signal sequence of the human ATX gene was truncated and replaced with sequence of insect cell secretion signal within expression vector. In addition, codons for six histidines were added to the C-termini of 120kDa ATX cDNA construct. A simple purification scheme utilizing two-step affinity column chromatography was designed to purify His-ATX to homogeneity from the culture supernatant of transfected insect cells. Homogenous His-ATX was detected and isolated from the concentrated insect cell medium using concanavalin A agarose and nickel affinity chromatography. Purified His-ATX was in full length with ATX capacity. A combination of this expression system and purification scheme would be useful for production and purification of high-quality functional ATX for research and practical application of multiple functional motogen, ATX/NPP-2.

• International Journal of Industrial Entomology and Biomaterials
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• v.29 no.2
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• pp.207-213
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• 2014

The structural proteins of classical swine fever virus (CSFV) consist of nucleocapsid protein C and envelope glycoprotein $E^{rns}$ (E0), E1 and E2. Among them, E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. In this study, to determine the optimal expression conditions of glycoprotein E2 using baculovirus system, we investigated the influence of insect cells and media to the expression of recombinant E2. Recombinant virus containing glycoprotein E2 coding gene was constructed with bApGOZA DNA. Expression of the glycoprotein E2 was analyzed by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. Expression of glycoprotein E2 in Sf21 cells was first observed after 3 days and reached a maximum on the 5th day after infection. Furthermore, the highest levels of glycoprotein E2 expression were observed at multiplicity of infection (MOI) of 5. When three different insect cell lines (Sf21, High-Five and Se301) were tested, High-Five cells showed the highest production. In addition, four different serum-free and serum-supplemented media, respectively, were tested for the expression of glycoprotein E2 and the budded virus (BV) titers. As a result, serum-supplemented medium provided the best conditions for protein production and the BV yield.

• The Plant Pathology Journal
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• v.37 no.5
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• pp.437-445
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• 2021

Tilletia laevis Kuhn (syn. Tilletia foetida (Wallr.) Liro.) causes wheat common bunt, which is one of the most devastating plant diseases in the world. Common bunt can result in a reduction of 80% or even a total loss of wheat production. In this study, the characteristics of T. laevis infection in compatible wheat plants were defined based on the combination of scanning electron microscopy, transmission electron microscopy and laser scanning confocal microscopy. We found T. laevis could lead to the abnormal growth of wheat tissues and cells, such as leakage of chloroplasts, deformities, disordered arrangements of mesophyll cells and also thickening of the cell wall of mesophyll cells in leaf tissue. What's more, T. laevis teliospores were found in the roots, stems, flag leaves, and glumes of infected wheat plants instead of just in the ovaries, as previously reported. The abnormal characteristics caused by T. laevis may be used for early detection of this pathogen instead of molecular markers in addition to providing theoretical insights into T. laevis and wheat interactions for breeding of common bunt resistance.

• International Journal of Industrial Entomology and Biomaterials
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• v.24 no.1
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• pp.37-40
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• 2012

The apidaecins are highly active against Gram-negative bacteria. Here, we show the expression and antibacterial activity of the bumblebee, Bombus ignitus, apidaecin. We PCR-amplified 51 bp of the active domain sequence of the B. ignitus apidaecin gene and expressed the recombinant B. ignitus apidaecin active domain in baculovirus-infected insect cells. The recombinant B. ignitus apidaecin active domain shows bactericidal activity against Gram-negative bacteria, including Pseudomonas tolaasii, a serious pathogen in cultivated mushrooms, but not Gram-positive bacteria. This result suggests that the active domain of the B. ignitus apidaecin is a potential antibacterial agent for the control of bacterial brown blotch diseases.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.5
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• pp.352-355
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• 2004

Heliothis zea (HzAM1) insect cells were immobilized in microspheres by sodium-cellulosesulfate (NaCS) and polydiallyldimethylammoniumchloride (PDADMAC). The highest HzAMl cell density was $7.5{\times}10^7$ cells/mL in the microspheres. After infection of the immobilized cells by Heliothis zea single nuclear polyhedrosis virus (HzSNPV), the highest concentration of HzSNPV (polyhedral inclusion bodies: PIBs) produced was $2.87{\times}10^{10}$ PIBs/mL in the microspheres.

• Biotechnology and Bioprocess Engineering:BBE
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• v.2 no.2
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• pp.113-116
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• 1997

The hydrogel process is a different form of ultrafiltration and has been used to separate biological molecules. In this study, the gel pore size was predicted by pulse NMR technique and neural network using a database obtained from gel filtration chromatography and diffusion experiment. Recombinant alkaline phosphatase expressed in insect cells was concentratred 1.5 times by hydrogel ultrafiltration by swelling at 2$0^{\circ}C$ and collapsing at 35$^{\circ}C$ at 53-65% separation efficiency and 78-83% enzyme recovery. Wild and recombinant Autographa californica unclear polyhedrosis viruses (AcNPV) were also concentrated 1.4 and 1.6 times of the feed solution at 48.5 and 60.0% separation efficiency, respectively Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of AcNPV and recombinant proteins from insect cells.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.853-857
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• 2001

The effect of silkworm hemolymph on the inhibition of baculovirus-induced insect cell apoptosis was quantitatively investigated using a flow cytometric analysis. Spodoptera frugiperda (Sf9) cell and Autographa californica nuclear polyhedrous virus (AcNPV) were used as a model for insect cell and baculovirus in this study, respectively. Compared with a mammalian cell cycle, the fraction of G1 cells was relatively small in the Sf9 cell cycle. Silkworm hemolymph did not affect the Sf9 cell-cycle distribution before the baculovirus infection. However, the fraction of cells which are not in the sub-G1 phase remained at a high level for 3 days after the infection in the medium without silkworm hemolymph, while it remained at a high level for 7 days after the infection in the medium supplemented with silkworm hemolymph. The fractions of apoptotic cells in the sub-G1 phase were $4.7\%$, and 4 days after infection, $22.7\%$, in the media with and without silkworm hemolymph, respectively.