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PubMed1996

A cyanogenic glycoside from Canthium schimperianum.

A new cyanogenic glycoside esterified with an iridoid glycoside, 2R-[(2-methoxybenzoylgenoposidyl)-5-O-beta-D-apiofuranosyl-( 1-->6)-beta-glucopyranosyloxy]-2-phenyl acetonitrile, was isolated from the seeds of Canthium schimperianum and identified from its spectroscopic data. The new compound showed weak growth retarding activity towards neonate larvae of Spodoptera littoralis (ED50 8580 ppm) compared to the co-occurring hydroxycinnamic acids, 3,4-dicaffeoylquinic acid (ED50 550 ppm) and 3,5-dicaffeoylquinic acid (ED50 520 ppm).

Animals↗
PubMed1994

Baculovirus expression of non-structural protein NS2 and core protein VP7 of African horsesickness virus serotype 3 and their use as antigens in an indirect ELISA.

Non-structural protein NS2 and core protein VP7 of African horsesickness virus serotype 3 (AHSV3) were expressed in Spodoptera frugiperda cells by recombinant baculoviruses containing the relevant genes. These proteins were purified and analysed by polyacrylamide gel electrophoresis and Western blot. NS2 and VP7 were used separately as antigens in an indirect ELISA for the detection of AHSV antibodies. Both antigens cross-reacted with hyperimmune guinea-pig antisera to infected cell lysates of all nine known AHSV serotypes and to antisera obtained from horses immunized with attenuated virus of seven AHSV serotypes.

African Horse Sickness Virus↗
PubMed1994

Production of measles nucleoprotein in different expression systems and its use as a diagnostic reagent.

Measles nucleoprotein has been successfully expressed in three different hosts, bacterial (Escherichia coli BL21 DE3), insect (Spodoptera frugiperda; Sf9) and mammalian (primary human fibroblasts) cells, each producing an antigenic protein of M(r) 60 kDa. The nucleoprotein produced in all three hosts was used in an ELISA for the detection of antibodies to measles virus in a cohort of haemagglutinin-positive or -negative human sera. Data produced from baculovirus and adenovirus-based antigens indicated that there was good correlation between the ELISA results and previous haemagglutination inhibition test data, and there was little background interference by cellular proteins or the development of false positive or negative results. The assay was rapid as it could be carried out in under 4 h, sensitive as the sera could be diluted by at least 1000-fold, and versatile as both IgG and IgM could be detected and differentiated.

Animals↗
PubMed1995

A diagnostic immunoassay for Newcastle disease virus based on the nucleocapsid protein expressed by a recombinant baculovirus.

A recombinant baculovirus containing a cDNA clone encoding the nucleocapsid (NP) protein of Newcastle disease virus (strain Ulster 2C) has been used to infect insect cells (Spodoptera frugiperda). High levels of overexpressed NP protein were observed, comprising up to 40% of total cellular protein, which were subsequently shown to be antigenic. Nucleoprotein derived from the crude soluble lysate of infected insect cells has been used in an indirect ELISA to detect the presence of anti-NDV antibodies in a cohort of chicken sera. Data produced from these tests indicated a good correlation between ELISA titre and haemagglutination inhibition test data. The test was not affected by interference from background cellular proteins nor by cross-reactivity with non-NDV poultry pathogens. Additionally, the test did not generate false-positive readings.

Animals↗
PubMed1996

Dengue virus envelope glycoprotein can be secreted from insect cells as a fusion with the maltose-binding protein.

The maltose-binding protein (MalE) contains a signal sequence which allows its translocation in the periplasm of prokaryotic microorganisms. In this study, MalE was produced in Spodoptera frugiperda (Sf9) lepidopterian cells using the baculovirus expression system. The secretion of MalE, following cleavage of its signal sequence, to the supernatant fluid of recombinant baculovirus-infected Sf9 cells and its affinity for maltodextrin polymers allowed recovery of significant amounts (> or = 10 micrograms per 10(6) cells) of highly purified protein. The gene encoding the envelope glycoprotein E of the dengue (DEN) type 2 virus deleted of its C-terminal 102 amino acids (D2E delta 102) was fused to the MalE gene. The resulting hybrid MalE-D2E delta 102 glycoprotein was processed through the Golgi network of Sf9 cells and was secreted. It was retained on a maltodextrin column and was eluted with maltose. Antigenic and immunogenic properties dependent on the three-dimensional structure in the native E protein were preserved in the recombinant MalE-D2E delta 102 protein. Thus MalE with its signal sequence may be used as a carrier protein for production in the baculovirus system and purification of proteins which require transportation through intracellular compartments for correct folding and processing.

Animals↗
PubMed1996

Green fluorescent protein as a tool for screening recombinant baculoviruses.

The gene encoding the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, ligated to the honeybee melittin signal peptide-encoding sequence, was inserted under transcriptional control of the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant green fluorescent protein was identified by SDS-PAGE gel electrophoresis followed by Coomassie blue staining of lysates from the recombinant baculovirus infected insect cells. Emission and excitation scanning of the recombinant baculovirus infected insect cells gave an emission maximum of 509 nm and excitation maximum of 398 nm. The GFP protein expressed was also detected in infected insect cells by a flow cytometer analysis.

Animals↗
PubMed1996

Expression and purification of the seven nonstructural proteins of the flavivirus Kunjin in the E. coli and the baculovirus expression systems.

All seven nonstructural (ns) proteins of the flavivirus Kunjin (KUN) ranging from NS1 to NS5 were expressed either alone or as fusion proteins with Glutathione-S-transferase (GST). High level expression of recombinant proteins was achieved in Spodoptera frugiperda (Sf9) cells using the baculovirus expression system in contrast to the low level of expression in E. coli. The order of the level of expression of the recombinant fusion proteins per 4 x 10(7) Sf9 cells was: GST-NS5 (yields approximately 4-5 mg) > GST-delta NS3 (approximately 1-2 mg) > GST-4A (approximately 1 mg) > GST-2B (approximately 0.5-1 mg) > GST-2A (approximately 0.5 mg) > GST-4B (approximately 0.1-0.2 mg). NS1 protein was expressed in a native form at the level of approximately 2-4 mg per 4 x 10(7) Sf9 cells. All the GST-fusion proteins were purified by adsorption on Glutathione Sepharose (GS) beads from solubilized lysates of Sf9 cells infected with the recombinant baculoviruses, or of E. coli cultures transformed with the expression plasmid and induced with IPTG. Only delta NS3 protein was recovered intact by removing GST from the fusion protein by digestion with Factor Xa protease. Attempts to cleave off the GST moiety from all the other purified recombinant proteins resulted either in inefficient cleavage or in degradation of the proteins. No GST-NS5 but from 20 to 50% of the purified GST-NS2A, GST-NS2B, GST-delta NS3, GST-NS4A, and GST-NS4B was eluted off the GS beads by adding glutathione. Thus, KUN purified recombinant proteins, either in eluted form or while immobilized on GS beads, could be used to raise monospecific antibodies, to perform functional assays or to participate in protein-protein or RNA-protein binding reactions.

Animals↗
PubMed1995

Intracellular signal transduction of PBAN action in lepidopteran insects: inhibition of sex pheromone production by compactin, an HMG CoA reductase inhibitor.

Pheromone biosynthesis activating neuropeptide (PBAN) regulates sex pheromone production in the pheromone glands of many species of female moths. In order to probe the biochemical steps as well as underlying mechanisms regulated by PBAN, we have tested the effect of chemicals on sex pheromone production by using an in vitro assay. Among the chemicals we tested here, compactin, a specific 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, clearly inhibited the pheromone biosynthesis in the silkworm, Bombyx mori, and the common cutworm, Spodoptera litura. Since the activation of HMG CoA reductase occurs by dephosphorylation mediated by a specific phosphatase and the biochemical step regulated by PBAN in bombykol biosynthesis is similar to the one catalyzed by HMG-CoA reductase in cholesterol biosynthesis, the present results support the idea that phosphoprotein phosphatase has a significant role to regulate bombykol production in the intracellular transduction of PBAN action in B. mori.

Amino Acid Sequence↗
PubMed1995

Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression.

Synthetic genes encoding the 146 and 155 amino acid forms of human basic fibroblast growth factor (bFGF) were constructed with codon usage biased towards the polyhedrin-encoding gene of Autographa californica nuclear polyhedrosis virus (AcNPV). Expression of both bFGF genes in Spodoptera frugiperda (SF-21) suspension cell culture using a recombinant baculovirus yielded approximately 2.5 mg of mitogenically fully active protein per 10(9) cells following heparin-affinity chromatography. To improve translational efficiency, the Kozak consensus sequence was introduced and it was found that neither the replacement of a pyrimidine by a purine at position -3, nor the nature of the base at position +4 had any noticeable effect on the final levels of bFGF expression in SF-21 cells. The bases at these critical points in the consensus do not therefore play a major role in expression levels of the bFGF synthetic genes. The two synthetic genes were also expressed in Escherichia coli as native proteins using the T7 expression system. 5 mg of mitogenically fully active bFGF were obtained from 1 l of bacterial culture. Both insect cell- and E. coli-derived bFGF were equally mitogenic for Swiss 3T3 fibroblasts.

3T3 Cells↗
PubMed1995

Physiological and environmental factors affecting the growth of insect cells and infection with baculovirus.

Insect cell growth can be significantly improved by close attention to the conditions used in the inoculum stages. Initial cell concentration, spent medium carry over and inoculum phase withdrawal significantly influenced the growth kinetics of Spodoptera frugiperda (Sf9) cells. The percentage of cells infected with wild and recombinant baculovirus AcNPV and (in the later case) the beta-galactosidase yield in fresh medium was appreciably affected by the stage of the growth curve that cells were in when infected and by the multiplicity of infection (MOI). However, the cell density at the time of infection and the medium condition showed little direct influence on infectivity. There may, however, be an indirect influence in that these factors determine the relative distribution of cells in the cell cycle. The infectivity is then in turn affected by the relative frequency of cells in the G1, S and G2/M phases. Insect cell specific oxygen uptake rates (1.3-3.4 x 10(-17) mol per cell per s) were essentially similar to or less than those measured for hybridoma cells. However, when Sf9 cells were infected with baculovirus, the specific oxygen uptake rate increased by up to 40%.

Animals↗
PubMed1995

Production and purification of recombinant African swine fever virus attachment protein p12.

The conditions for cultivation of Spodoptera frugiperda (Sf9) insect cells for production of recombinant baculoviruses have been studied, to scale-up and improve the efficiency of the process for production of the African swine fever virus attachment protein p12 in the baculovirus expression system. It was shown that the total virus and recombinant protein production in insect cells infected with the Acp12 recombinant baculovirus were slightly dependent on cell density, but largely dependent on the serum concentration, in the case of suspended cells, but not in static monolayer cultures. The yield of recombinant protein p12 exceeded 50 mg per 1 of 2 x 10(9) cells, representing more than 10% of total cell proteins, a level > 20-fold higher than that observed with other eukaryotic expression systems. The presence of p12 in the cytoplasmic fraction of infected cells has allowed the purification of the protein by a simple two-step procedure of aqueous phase partition and octyl-glucoside solubilization. The recombinant protein p12 was able to inhibit, in a dose-dependent manner, the African swine fever virus production in swine macrophages infected with a number of different virus isolates, including attenuated, virulent, highly passaged on tissue culture, and non-haemadsorbing strains, indicating a fundamental role for p12 in the early interaction of the virus with the natural target cell receptors. However, pigs immunized with purified recombinant p12 did not develop protective immunity against African swine fever.

African Swine Fever Virus↗
PubMed1996

Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector.

Bacillus thuringiensis (Bt) delta-endotoxins are safe biological insecticidal proteins whose usefulness has long been recognized. The first commercialized Bt insecticidal formulations were composed of spore-crystal preparations derived from wild-type strains. These products generally have a limited insecticidal host range and several genetically modified strains have, therefore, been constructed using transformation procedures. However, addition of a new delta-endotoxin gene to strains already harboring other delta-endotoxin genes often resulted in broader-spectrum but less potent products because they produced significantly less of each of the crystal proteins. We report expression of the coding sequence of the sporulation specific cryIC gene from the non-sporulation-dependent cryIIIA promoter. Large amounts of CryIC accumulated in various Bt strains with different genetic backgrounds. Sporulation deficient Spo0A mutants, acrystalliferous derivatives and wild-type Bt strains expressing the engineered cryIII-cryIC gene were obtained. Introduction of the cryIII-cryIC gene whose product is highly active against Spodoptera littoralis into the Kto strain harboring the cryIA(c) gene active against Ostrinia nubilalis resulted in the construction of a new strain with increased potency and broader activity spectrum than the parent strain. Large amounts of each toxin were produced and the expression of the two genes seemed to be summed, presumably because the expression systems of the two genes are different. The plasmid shuttle vector used to introduce the cryIII-cryIC gene into the different Bt hosts utilizes the specific resolution site of transposon Tn4430 to enable construction of recombinant Bt strains that are free of foreign non-Bt DNA. This should facilitate the approval and acceptance for environmental release of the insecticidal recombinant products.

Animals↗
PubMed1996

Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: large scale production of the E1 protein.

The two envelope glycoproteins of rubella virus (RV), E1 of 58 kDa and E2 of 42-47 kDa, were individually expressed in lepidopteran Spodoptera frugiperda as well as in Trichoplusia ni insect cells using baculovirus vectors. The authentic signal sequences of E1 and E2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E1 and E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes. Synthesis of the recombinant proteins in the absence and presence of tunicamycin revealed that both E1 and E2 were glycosylated with apparent molecular weights of 52 kDa and 37 kDa, respectively. Recombinant E2 appeared to be partially secreted, whereas E1 was essentially found inside the infected insect cell. The E1 protein was produced in large scale using a 10-1 bioreactor and serum-free medium (SFM). Purification of the recombinant protein product was performed from cytoplasmic extracts by ammonium sulphate precipitation followed by Concanavalin A affinity chromatography. This type of purified recombinant viral glycoproteins may be useful not only in diagnostic medicine or for immunization, but should enable studies designed to solve the structure of the virus particle.

Animals↗
PubMed1995

Expression of Marek's disease virus (MDV) serotype 2 gene which has partial homology with MDV serotype 1 pp38 gene.

We constructed the recombinant baculovirus expressing the gene of non-pathogenic Marek's disease virus (MDV) serotype 2 (MDV2) which encodes a polypeptide with partial homology to MDV serotype 1 (MDV1) pp38, an antigen associated with transformed cells. The recombinant MDV2 protein was detected as a band of 32 kDa in immunoblot analysis with MDV2-infected chicken serum. Mouse serum against insect Spodoptera frugiperda cells infected with the recombinant baculovirus immunoprecipitated a 38 kDa molecule from the lysate of MDV2-infected chicken embryo fibroblasts (CEFs) but did not immunoprecipitate the MDV1 pp38 from the lysate of MDV1-infected CEFs. This result indicates that the recombinant MDV2 protein has no epitopes shared with the MDV1 pp38.

Animals↗
PubMed1995

Preparation of monoclonal antibodies against Marek's disease virus serotype 1 glycoprotein D expressed by a recombinant baculovirus.

A recombinant baculovirus, the genome of which contains DNA encoding Marek's disease virus serotype 1 (MDV1) homolog of glycoprotein D (gD) of herpes simplex virus under the polyhedrin promoter was constructed and designated rAcMDV1gD. Five monoclonal antibodies (MAbs) which recognize the MDV1 homolog of gD (MDV1 gD) in Spodoptera frugiperda cells infected with rAcMDV1gD were prepared. The MAbs reacted with proteins ranging from 52 to 49 kDa in rAcMDV1gD-infected cell lysates by immunoblot analysis. These molecular weights were coincident with molecular weights predicted from the open reading frame of MDV1 gD. By ELISA additivity test, the 5 MAbs were divided into 3 groups which seemed to recognize 3 different epitopes. In addition, all of the 5 MAbs were reactive with chick embryo fibroblasts (CEFs) expressing MDV1 gD. The MAbs are considered to be useful to study the role of MDV1 gD in MDV1 infection.

Animals↗
PubMed1995

Immunogenic properties of rabbit haemorrhagic disease virus structural protein VP60 expressed by a recombinant baculovirus: an efficient vaccine.

We have constructed a recombinant baculovirus containing the gene encoding the structural protein VP60 from the Spanish field isolate AST/89 of rabbit haemorrhagic disease virus (RHDV). Infection of cultured Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of high yields of VP60 protein which did not seem to assemble to form virus like particles, but was antigenically similar to the corresponding viral protein obtained from purified virions. A VP60-dose study showed that the recombinant protein was able to elicit a protective response in rabbits against a nasal challenge with 100 LD50 of RHDV. The effective dose able to protect 50% of the animals in the absence of adjuvant was found to be 10-25 micrograms of recombinant VP60.

Animals↗
PubMed1995

Expression and characterization of the zeta 1 subunit of the N-methyl-D-aspartate (NMDA) receptor channel in a baculovirus system.

Using a baculovirus expression vector system, the zeta 1 subunit of the mouse N-methyl-D-aspartate (NMDA) receptor channel was expressed in Spodoptera frugiperda insect cells. The peptide corresponding to the C-terminus of the zeta 1 subunit was synthesized by using the multiple antigen peptide (MAP) system, and an antibody to the synthetic peptide was produced. Immunoblotting using the newly developed antibody revealed the major 122-kDa and the minor 104-kDa protein bands. The effect of tunicamycin on the immunoblots and [35S]methionine/[35S]cysteine metabolic radiolabeling suggested that the two bands corresponded to glycosylated and non-N-glycosylated forms, respectively. Membranes prepared from insect cells infected with the recombinant virus had the binding activity of antagonist ligand 5,7-[3-3H]dichlorokynurenate (DCKA) of a glycine recognition domain of the receptor. Both immunofluorescence labeling and the [3H]DCKA binding assays also showed a greater level of expression (Bmax = 51 pmol/mg protein) in the insect cells. The ligand binding characteristics of the receptors expressed in insect cells suggested that the single zeta 1 subunit protein has glycine antagonist binding properties comparable to those of the native NMDA receptor channels. The lack of DCKA-binding activity of the non-N-glycosylated NMDA receptor expressed in the presence of tunicamycin suggested that N-linked oligosaccharide is essentially required for expression of a functional receptor in insect cells. This is the first report describing the importance of N-glycosylation for the acquisition of ligand binding to NMDA receptor channel subunit protein.

Animals↗
PubMed1996

Expression, purification and characterization of recombinant C. Elegans UNC-18.

The Caenorhabditis elegans unc-18-encoded protein (UNC-18) is implicated in the processes of vesicle targeting, docking, and/or fusion. To further characterize the properties of this important neural protein, we expressed it at a high level in Spodoptera frugiperda Sf21 cells using a baculovirus expressing system. A cDNA containing the coding sequence for UNC-18 was inserted into the transfer vector pBlueBac to yield the recombinant virus pAcNPV/unc-18. At maximal expression, the recombinant virus produces a protein of 67 kDa, which constitutes about one-third of total cell protein. The UNC-18 protein was highly purified and its biochemical and functional properties were assessed. The protein is globular with an isoelectric point of 6.95. Circular dichroism spectroscopy indicated that the alpha-helix and beta-sheet account for 10.0 and 59.0%, respectively. Immunolabeling the Sf21 cells expressing UNC-18 showed that the expressed UNC-18 is predominantly localized in the cytoplasm as a soluble monomer. The protein is phosphorylated by protein kinase C and binds to the recombinant C. elegans syntaxin in vitro. These findings suggest that in vesicle traffic UNC-18 is a regulator factor associated with the plasma membrane through syntaxin, although intrinsically cytoplasmic.

Animals↗