Nuclear Polyhedrosis Virus Research Articles

517. Type 1 Rep52 Is Superior to Authentic Rep52 for Producing Recombinant Adeno-Associated Virus Type 5 in Insect Cells

Top of pageAbstract The recently described method for producing recombinant adeno-associated virus (rAAV) type 2 in insect cells facilitates scale-up by using suspension cell cultures and baculovirus expression vectors (Hum Gene Ther 13:1935-1943, 2002). AAV5 is one of the most divergent dependovirus characterized and has been shown to utilize different receptors than AAV2 for example, and has demonstrated different tissue tropism from other serotype rAAVs. In addition to vectors derived from serotype 2, other serotypes constitute a vector set from which an optimal one can be selected for specific applications. Thus, we established a method to generate rAAV5 in insect cells. The current process requires triple infection with recombinant baculoviruses that provide the following: 1. AAV structural proteins that form the virus capsid (VP 1, 2, 3); 2. two of the AAV non-structural proteins for replication and encapsidation (Rep 78 and Rep 52); and 3. the AAV vector DNA which contain the gene of interest flanked by the AAV origins of replication (ITRs). Compared to other rAAV serotypes produced in insect cells, the rAAV5 yield per cell was substantially lower and we tested some modifications of the system. The initial Rep baculovirus drove type 5 Rep72 expression with a truncated promoter for the immediate-early 1 gene of Orgyia pseudotsugata nuclear polyhedrosis virus. The titers of the Rep baculovirus were relatively lower than others. We constructed Rep baculoviruses with a series of truncated p10 promoters for Rep78 and selected one that could produce rAAV5 at a high titer and propagate well. Rep52 or small Rep protein packages AAV genome into preformed empty capsids. We examined Rep52 of other serotypes, 1, 2, 3, 4 for generation of rAAV5 particles. The titer of rAAV5-GFP produced with type 1, 2, 3, or 4 small Rep was 56,000|[plusmn]|3,200, 41,000|[plusmn]|18,900, 42,000|[plusmn]|7,300, or 39,000|[plusmn]|3,500 particles per Sf9 cell while rAAV5-GFP produced by authentic Rep52 was 13,500|[plusmn]|3,200. The rAAV5-GFP produced with either serotype small Rep has an equal ratio of nucleic acid to capsid protein assessed by real-time PCR quantification and silver staining of purified rAAV5 particles. Also, rAAV5-GFP produced with either combination of large and small Rep proteins, transduced the simian Cos cell line with similar efficiency. The analysis by cesium density gradient of insect-cell lysates indicated that approximately 50% of all capsids contained the vector genome. These results indicate that heteroserotypic small Rep polypeptide is able to substitute for AAV5 small Rep and package AAV vector genome with type 5 in Sf9 cells and the new Rep baculovirus expressing type 5 Rep78 under the control of a truncated p10 promoter and type 1 Rep52 will contribute to the development of more efficient production of rAAV5 in insect cells.

Improving the efficacy of nuclear polyhedrosis virus and Bacillus thuringiensis against Helicoverpa spp. with ultra-violet light protected petroleum spray oils on cotton crops in Australia

Nuclear polyhedrosis virus (NPV) and Bacillus thuringiensis (Bt) are the most commonly used biopesticides for the control of Helicoverpa spp. larvae on cotton crops in Australia. The performance of NPV and Bt against Helicoverpa spp. larvae on cotton crops, is inconsistent and at times totally unsatisfactory against high densities of Helicoverpa spp. larvae. We determined the effect of mixing petroleum spray oils, containing ultra-violet light absorbing compounds, with NPV and Bt for efficacy against Helicoverpa spp. larvae, levels of cotton plant damage, and persistence of efficacy. The study showed that the efficacy and persistence of NPV and Bt were increased when mixed with petroleum spray oil (PSO – Canopy®) at the rate of 2% (v/v). In the field experiments, mixing NPV with 1 and 2% (v/v) PSO, increased Helicoverpa spp. mortality from 25.9 to 31.5 and 44.8%, respectively. Similarly, the mortality caused by Bt, when mixed with 1 and 2% (v/v) PSO, was increased from 31.5 to 36.0 and 48.2%, respectively. In addition, 1 and 2% PSO mixtures with NPV increased persistence of efficacy from 1.1 to 1.6 and 2.5 days, respectively, whilst persistence of Bt was increased from 1.5 to 1.8 and 2.6 days, respectively. In another study using potted cotton plants, in which the plants were left outdoors throughout the study, the average NPV induced mortality of first instar Helicoverpa larvae was increased from 20.9% to 35.9 and 43.4% by 1 and 2% (v/v) PSO, respectively. Persistence of NPV efficacy was enhanced by 2 and 3.1 times by 1 and 2% (v/v) PSO, respectively. Similarly, Bt induced mortality of Helicoverpa larvae was increased by 1 and 2% PSO from 68.1 to 78.8 and 83.2%, respectively, and the persistence of Bt efficacy was enhanced 1.3 – 2.0 times, respectively. In a mesh house study, young cotton plants, treated with a PSO/biopesticide mixture, suffered less leaf damage than cotton plants treated with the biopesticides alone. In conclusion, the results of this study showed synergies from the combined use of UV protected PSO and NPV or Bt, against Helicoverpa spp. larvae on cotton. Such a biopesticide-PSO combinations could be a useful tool for IPM program in cotton.

Microarray and RT-PCR screening for white spot syndrome virus immediate-early genes in cycloheximide-treated shrimp

Here, we report for the first time the successful use of cycloheximide (CHX) as an inhibitor to block de novo viral protein synthesis during WSSV (white spot syndrome virus) infection. Sixty candidate IE (immediate-early) genes were identified using a global analysis microarray technique. RT-PCR showed that the genes corresponding to ORF126, ORF242 and ORF418 in the Taiwan isolate were consistently CHX-insensitive, and these genes were designated ie1, ie2 and ie3, respectively . The sequences for these IE genes also appear in the two other WSSV isolates that have been sequenced. Three corresponding ORFs were identified in the China WSSV isolate, but only an ORF corresponding to ie1 was predicted in the Thailand isolate. In a promoter activity assay in Sf9 insect cells using EGFP (enhanced green fluorescence protein) as a reporter, ie1 showed very strong promoter activity, producing higher EGFP signals than the insect Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) ie2 promoter.

Molecular Cloning of Mouse Type 2 and Type 3 Inositol 1,4,5-Trisphosphate Receptors and Identification of a Novel Type 2 Receptor Splice Variant

We isolated cDNAs encoding type 2 and type 3 inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R2 and IP(3)R3, respectively) from mouse lung and found a novel alternative splicing segment, SI(m2), at 176-208 of IP(3)R2. The long form (IP(3)R2 SI(m2)(+)) was dominant, but the short form (IP(3)R2 SI(m2)(-)) was detected in all tissues examined. IP(3)R2 SI(m2)(-) has neither IP(3) binding activity nor Ca(2+) releasing activity. In addition to its reticular distribution, IP(3)R2 SI(m2)(+) is present in the form of clusters in the endoplasmic reticulum of resting COS-7 cells, and after ATP or Ca(2+) ionophore stimulation, most of the IP(3)R2 SI(m2)(+) is in clusters. IP(3)R3 is localized uniformly on the endoplasmic reticulum of resting cells and forms clusters after ATP or Ca(2+) ionophore stimulation. IP(3)R2 SI(m2)(-) does not form clusters in either resting or stimulated cells. IP(3) binding-deficient site-directed mutants of IP(3)R2 SI(m2)(+) and IP(3)R3 fail to form clusters, indicating that IP(3) binding is involved in the cluster formation by these isoforms. Coexpression of IP(3)R2 SI(m2)(-) prevents stimulus-induced IP(3)R clustering, suggesting that IP(3)R2 SI(m2)(-) functions as a negative coordinator of stimulus-induced IP(3)R clustering. Expression of IP(3)R2 SI(m2)(-) in CHO-K1 cells significantly reduced ATP-induced Ca(2+) entry, but not Ca(2+) release, suggesting that the novel splice variant of IP(3)R2 specifically influences the dynamics of the sustained phase of Ca(2+) signals.

The Function of Envelope Protein P74 from Autographa californica Multiple Nucleopolyhedrovirus in Primary Infection to Host

This research investigated the function of envelope protein P74 of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) in primary infection to host. A p74-inactivation recombinant baculovirus, rAc-gfp(Delta) p74, was constructed by inserting gfp driven by AcMNPV polyhedrin promoter into the p74 locus of AcMNPV genome. Bioassays showed that the P74-null occlusion bodies (OBs) failed to infect its natural host larvae, Spodoptera exigua, per os, while the p74-null budded virus (BVs) could infect host larvae by injection. However, its inability for oral infectivity was rescued by a mixed infection with wild-type OBs or with the purified P74 protein expressed in Spodoptera frugiperda Sf-9 cells, and the P74 protein rescue was in a dosage-dependent manner. The 50% lethal dosage (LD50) value of a P74 overexpression recombinant virus, rAc-p74(++)-polh+, which contained two copies of p74 gene, was not significantly different from that of wild-type virus. One-step growth curve assays of viruses suggested that BV production from cells infected with p74-null virus was similar to that from cells infected with wild-type virus or the P74 overexpression virus. ELISA analysis indicated that P74 protein could bind its host brush border membrane vesicles (BBMV) efficiently with saturation, but it could only bind its sensitive midgut BBMV specifically. In vitro pull-down assay showed that a protein of approximately 35 kDa in the BBMV was involved in the specific binding. These results demonstrated that the P74 protein is essential for oral infectivity of occlusion-derived virus (ODV) and plays a role in midgut attachment and fusion.

Improved artificial diet for mass rearing of the tobacco caterpillar, Spodoptera litura (Lepidoptera: Noctuidae)

An artificial diet for mass rearing of the tobacco caterpillar Spodoptera litura (Fabricius) from the neonate to adult stage was developed at 27±1°C, 65±5% RH and 16:8 h scoto/photo-phase regime. The diet ingredients consisted of wheat germ (26.0 g), kidney bean flour (51.3 g), chickpea flour (56.0 g), yeast powder (31.6 g), casein (15.2 g), l-ascorbic acid (3.2 g), cholesterol (0.5 g), two multivitamin multimineral capsules, one vitamin E capsule, ABDEC drops (2 ml), castor oil (1 ml), methyl- p -hydroxybenzoate (1.8 g), sorbic acid (1.3 g), streptomycin sulphate (0.25 g), formaldehyde solution (2 ml), agar–agar (16.4 g) and 820 ml distilled water. The new artificial diet successfully supported the growth and development for more than ten generations with enhanced reproductive potential. The mean biological parameters based on rearing of ten continuous generations showed higher pupation (89.2%), emergence (97.2%), survival (86.6%) and fecundity (2486.2 eggs) as compared to the most preferred natural food, castor leaf whose respective recorded values are 80, 75, 60% and 480 eggs. The cost of 1 l of the diet was approximately US$ 2.00 only, on which 200 neonate larvae can be reared. The artificial diet developed is suitable for mass rearing S. litura successfully throughout the year for laboratory and field experimentation as well as for commercial production of nuclear polyhedrosis viruses (Sl NPV).

Involvement of the Toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus.

We have previously shown that mice inoculated intranasally with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus [AcNPV]) are protected from a lethal challenge by influenza virus. However, the precise mechanism of induction of this protective immune response by the AcNPV treatment remained unclear. Here we show that AcNPV activates immune cells via the Toll-like receptor 9 (TLR9)/MyD88-dependent signaling pathway. The production of inflammatory cytokines was severely reduced in peritoneal macrophages (PECs) and splenic CD11c(+) dendritic cells (DCs) derived from mice deficient in MyD88 or TLR9 after cultivation with AcNPV. In contrast, a significant amount of alpha interferon (IFN-alpha) was still detectable in the PECs and DCs of these mice after stimulation with AcNPV, suggesting that a TLR9/MyD88-independent signaling pathway might also participate in the production of IFN-alpha by AcNPV. Since previous work showed that TLR9 ligands include bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides, we also examined the effect of baculoviral DNA on the induction of innate immunity. Transfection of the murine macrophage cell line RAW264.7 with baculoviral DNA resulted in the production of the inflammatory cytokine, while the removal of envelope glycoproteins from viral particles, UV irradiation of the virus, and pretreatment with purified baculovirus envelope proteins or endosomal maturation inhibitors diminished the induction of the immune response by AcNPV. Together, these results indicate that the internalization of viral DNA via membrane fusion mediated by the viral envelope glycoprotein, as well as endosomal maturation, which releases the viral genome into TLR9-expressing cellular compartments, is necessary for the induction of the innate immune response by AcNPV.

Facile monitoring of baculovirus infection for foreign protein expression under very late polyhedrin promoter using green fluorescent protein reporter under early-to-late promoter

A recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV) was constructed so that the green fluorescent protein (GFP) was produced via the early-to-late (ETL) promoter. This enabled rapid monitoring of the infection of Sf-9 insect cells. Notably, GFP and its fluorescence appeared ∼18 h prior to proteins expressed using very late polyhedrin (Polh) promoter. It is anticipated that the use of GFP under the control of ETL promoter will facilitate vector construction, virus isolation, and titer determination.

Construction of a Transposon-mediated Baculovirus Vector Hanpvid and a New Cell Line for Expressing Barnase

In this study we developed the transposon-mediated shuttle vector 'Hanpvid', which composed of HaNPV (Heliothis armigera nuclear polyhedrosis virus) genomic DNA and a transposon cassette from Bacmid of Bac-to-Bac system. Hanpvid replicates in E. coli in the same way as Bacmid and retains infective function in cotton bollworm cells (Hz-AM1). Using Hanpvid we constructed a recombinant virus, which could infect Hz-AM1 cells and generate recombinant HaNPV (rHa-Bar) containing the barnase gene, a ribonuclease gene from Bacillus amyloliquefaciens. Since the expression vector carrying barnase gene cannot replicate in the absence of barstar, a specific inhibitor of barnase, we constructed a new cotton bollworm cell line (AM1-NB) using the marker rescue method. In AM1-NB barstar was integrated into the cellular chromosome to sustain the replication of rHa-Bar. To screen out recombinant HaNPV for potential use as biopesticide, Hz-AM1 and AM1-NB cell lines were infected with rHa-Bar, respectively. The results obtained indicate that Viral progenies in AM1-NB were 23 and 160 times greater than those in Hz-AM1 48 h and 72 h after infection, respectively. With additional insertion of the polyhedron gene from AcNPV (Autographa californica nuclear polyhedrosis virus) into the Hanpvid genome, rHa-Bar regained the polyhedron phenotype and its pest-killing rate greatly improved. Toxic analysis showed that the lethal dosages (LD(50)) and the lethal time(s) (LT(50)) of rHa-Bar were reduced by 20 % and 30 %, respectively, compared to wt-HaNPV in the third instar larvae of cotton bollworm. This study shows that in AM1-NB barnase can be effectively produced and used as pest-killing agent for the biological control of cotton pests.

Cloning and characterization of the G protein βγ subunits from Trichoplusia ni (High Five™ cells)

Baculoviral-mediated expression in insect cells has become a method of choice where high-level protein expression is desired and where expression in Escherichia coliform ( E. coli.) is unsuitable. Genes of interest are inserted into the baculoviral genome of Autographa californica nuclear polyhedrosis virus (AcNPV) under the extremely strong, but very late polyhedron gene (PolH). The preferred host lines are derived from Spodoptera frugiperda ( Sf9 or Sf21) or Tricoplusia ni (High Five™, Invitrogen). Viral expression in insect cells is commonly used in the signal transduction field, due to the more than satisfactory capacity to express membrane proteins. However, co-association and/or co-purification of contaminating endogenous host G protein subunits, for example, may potentially threaten the functional and structural homogeneity of membrane preparations. The undefined G protein composition is complicated by the limited sequence data of either the S. frugiperda or Tricoplusia ni genomes. Here we report the isolation of cDNAs encoding two members of the heterotrimeric G protein family, G β ( Tn-G β) and G γ ( Tn-G γ), from Tricoplusia ni. Tn-G β shares ∼90% amino acid sequence identity with G β from Drosophila melanogaster and 84% identity with mammalian G β (human G β1). Tn-G γ shares approximately 71% amino acid identity with D. melanogaster G γ1 and 42% identity with mammalian G γ (human G γ2). Tn-G βγ is also functionally similar to mammalian G β1 γ2 by virtue of their capacity to form a complex with mammalian G α subunits, support G-protein-dependent agonist binding to a mammalian G protein-coupled receptor ( β2-adrenergic receptor) and directly regulate effectors such as adenylyl cyclase.

Fluorescent differential display analysis of gene expression for NPV resistance in Bombyx mori L.

Using the fluorescent differential display (FDD) technique, we analysed the differential expression of genes related to BmNPV (Bombyx mori nuclear polyhedrosis virus) resistance. Silkworm strains used included a highly resistant strain named NB, a highly susceptible strain named 306 and a near-isogenic line named 306NNZZ. Two differential bands, G12 532 and G13 313 , were found linked to BmNPV resistance and were further identified. Band G12 532 was found in the midguts of the eighth generation of backcross (BC 8 ) of strain 306NNZZ but was not present in those of strain 306. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed that the band existed in BC 8 of strains 306NNZZ and NB while not in strain 306. Northern blot analysis showed that G12 532 expressed actively in BC 8 of strains 306NNZZ and NB while expressed inactively in strain 306. These results suggest that the G12 532 is a fragment of the gene related to silkworm's resistance to BmNPV disease. The sequence of G12 532 has an 85% similarity to sequences with GenBank accession numbers of AV398077 and AV398034 that are relative to BmNPV infection demonstrated by previous studies. Its coding region (37 amino acids) has 54% similarity with B. mori endonuclease and reverse transcriptase-like protein. Band G13 313 was appeared in the hemolymph of highly resistant strain NB and near isogenic line 306NNZZ in FDD, but was not found in highly susceptible strain 306, all of which were confirmed by RT-PCR and Northern blot analyses. All the experimental results obtained suggest that G13 313 is also a fragment of the gene related to BmNPV resistance. The sequence of band G13 313 does not have a high similarity to the above relative sequences in GenBank. It might be a new RNA fragment related to NPV resistance. Our study also showed that the FDD technique could be a promising approach in identifying novel genes while appropriate silkworm strains are used.

Characterization of a Second Arabidopsis thaliana Prolyl 4-Hydroxylase with Distinct Substrate Specificity

4-Hydroxyproline is found in collagens, collagen-like proteins, elastin, and the hypoxia-inducible transcription factor in animals and in many hydroxyproline-rich glycoproteins in plants. We report here on the cloning and characterization of a second plant P4H (prolyl 4-hydroxylase), At-P4H-2, from Arabidopsis thaliana. It consists of 299 amino acids and shows 33% sequence identity to the first characterized isoenzyme, At-P4H-1. A characteristic feature of the At-P4H-2 polypeptide is a 49-amino-acid C-terminal toxin homology domain with 6 cysteines that is not found in At-P4H-1 but is present in a putative rice P4H homologue. At-P4H-2 differed distinctly from At-P4H-1 in its substrate specificity. Recombinant At-P4H-2 hydroxylated poly(L-proline) and extensin and arabinogalactan-like peptides effectively but with much higher Km values than At-P4H-1, suggesting different roles for the two At-P4Hs in the plant cell. Unlike At-P4H-1, At-P4H-2 hydroxylated collagen-like peptides only very inefficiently and did not hydroxylate hypoxia-inducible transcription factor alpha-like peptides at all. All the peptides efficiently hydroxylated by At-P4H-2 had at least 3 consecutive prolines, suggesting that these may represent a minimum requirement for efficient hydroxylation by this isoenzyme. N-terminal sequencing of an extensin-like peptide SPPPVYKSPPPPVKHYSPPPV indicated that At-P4H-2 preferentially hydroxylated the 3rd proline in the C-terminal PPP triplet. The Km values of At-P4H-2 for the reaction cosubstrates Fe2+, 2-oxoglutarate, and ascorbate were similar to those of At-P4H-1 with the exception that the Km for iron was about 3-fold lower. Pyridine-2,4-dicarboxylate and pyridine-2,5-dicarboxylate, well known competitive inhibitors of the vertebrate P4Hs with respect to 2-oxoglutarate, were also competitive inhibitors of At-P4H-2 but with Ki values 5-100-fold higher than those of human type I collagen P4H. It thus seems that there are some distinct differences in the structure of the 2-oxoglutarate-binding site between At-P4H-2 and the animal collagen P4Hs.

Baculoviral polyhedrin-Bacillus thuringiensis toxin fusion protein: A protein-based bio-insecticide expressed inEscherichia coli

Previously, we found that baculoviral polyhedrin (Polh) used as a fusion partner for recombinant expression in Escherichia coli showed almost the same characteristics (rapid solubilization under alkaline conditions and specific degradation by specific alkaline proteases in insect midgut) as the native baculoviral Polh, and formed easily isolatable inclusion bodies. Here, Polh derived from the Autographa californica nuclear polyhedrosis virus (AcNPV) was fused with a Bacillus thuringiensis (Bt) toxin protein (truncated Cry1Ac having toxin region as a model Bt toxin) for the novel generation of a new bio-insecticide. The Polh-Cry1Ac fusion protein (approximately 99 kDa) was highly expressed (3.6-fold induction as compared to E. coli-derived single Cry1Ac (approximately 68 kDa)) as an insoluble inclusion body fraction in E. coli. Trypsin and alpha-chymotrypsin, which have similar properties to the insect midgut alkaline proteases, rapidly degraded the Polh portion in vitro, leaving only the toxic Cry1Ac protein behind. In vivo, the Polh-Cry1Ac fusion protein showed high insecticidal activity against the pest, Plutella xylostella. Because this novel bio-insecticide employs E. coli as the host, mass production at a low cost should be possible. Also, since this is a protein-based insecticide, living modified organism (LMO) issues such as environmental and ecological safety can be avoided.

High-level expression of recombinant 3AB1 non-structural protein from FMDV in insect larvae

For its potential usefulness in diagnosis, the non-structural protein 3AB1 from foot-and-mouth disease virus was expressed as a soluble protein by using Autographa californica nuclear polyhedrosis virus as a vector. The 3AB1 coding sequence was introduced into AcNPV genome via pBAcPAK3AB1 transfer vector to originate Ac3AB1 recombinant baculovirus of phenotype occ −. Rachiplusia nu larvae were injected with supernatants of Sf9 cells infected with Ac3AB1 and 5 days post-infection total protein extracts were obtained. An intense band of approximately 21.5 kDa was observed when total larvae extracts were SDS-PAGE resolved and the recombinant protein detected by an FMDV-infected guinea pig serum. ELISA tests and Western blot experiments were carried out using sera both from FMDV-infected cattle and from vaccinated animals. The recombinant protein was only recognized by sera from infected animals, suggesting that this method of production in insect larvae could be applied to an efficient mass production of proteins of diagnostic interest.

Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system

Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses.

Mass production of HzSNPV baculoviruses in immobilizedHeliothis zea (HzAM1) insect cell culture

Heliothis zea (HzAM1) insect cells were immobilized in microspheres by sodium-cellulosesulfate (NaCS) and polydiallyldimethylammoniumchloride (PDADMAC). The highest HzAMI cell density was 7.5×107 cells/mL in the microspheres. After infection of the immobilized cells byHeliothis zea single nuclear polyhedrosis virus (HzSNPV), the highest concentration of HzSNPV (polyhedral inclusion bodies: PIBs) produced was 2.87×1010 PIBs/mL in the microspheres.

Promoter and piggyBac activities within embryos of the potato tuber moth, Phthorimaea operculella, Zeller (Lepidoptera: Gelechiidae)

Potato production in tropical and subtropical countries suffers from damage caused by the potato tuber moth (PTM), Phthorimaea operculella. The aim of this research was the development of the components required for a germline transformation system for the PTM. We tested three components that are critical to genetic transformation systems for insects: promoter activity, marker gene expression, and transposable element function. We compared the transcriptional activities of five different promoters, hsp70, hsp82, actin5C, polyubiquitin and immediate early 1 gene ( ie1), within PTM embryos. The ie1 promoter, flanked by the hr5 enhancer element, showed a very high level of transcriptional activity compared to the other promoters. The fluorescence activity of EGFP was also determined and transient expression of EGFP was detected in 57% of injected embryos. The transpositional activity of the piggyBac transposable element was tested in an interplasmid transposition assay. The piggyBac element was shown to be mobile within the embryonic soma of the PTM with a transposition frequency of 4.2×10 −5 transpositions/donor plasmid. Incorporating a transactivator plasmid expressing the immediate early protein (IE1) from the Bombyx mori nuclear polyhedrosis virus enhanced the efficiency of piggyBac mobility.

Expression and one-step purification of bovine interleukin-21 (IL-21) in silkworms using a hybrid baculovirus expression system

A hybrid baculovirus, a hybrid of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of bovine interleukin-21 (IL-21) in silkworms. A recombinant hybrid baculovirus containing the full length of the cDNA of bovine interleukin-21 was constructed and used to infect silkworm larvae or silkmoth pupae. After the infection of the virus, bovine mature IL-21 was produced in the haemolymph or pupal cell lysates. A one-step purification of bovine mature IL-21 from haemolymph using a cation exchange column gave 0.5 mg. IL-21 from 30 ml haemolymph. The bovine IL-21 produced by silkworms strongly induced NK cell proliferation using a human NK cell-line, NK0, and enhanced the lymphokine activated killer (LAK) activity of bovine peripheral blood mononuclear cells.

The murine thymic stromal lymphopoietin receptor is partially expressed with an extracellular C-terminus

The gene encoding the murine thymic stromal lymphopoietin receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica Nuclear Polyhedrosis Virus and expressed with an N-terminal poly-histidine tag and a C-terminal FLAG-tag in the Spodoptera frugiperda insect cell line Sf9 during viral infection. Flow cytometer analysis of cells infected with the produced recombinant virus FastBacHisB-mδ1-FLAG demonstrated that a majority of the infected cells expressed the mTSLPR with an extracellular C-terminal end. A similar observation was noticed in COS cells transfected with pSVL-mTSLPR-FLAG. Immunoblotting with monoclonal anti-FLAG or anti-his antibodies indicated that the corresponding receptor protein migrated as an approximately 50 kDa protein. mTSLPR produced in presence of tunicamycin migrated with a molecular weight around 40 kDa. The genetically fused poly-histidine tag was also demonstrated to be functional using a Ni-NTA purification system, indicating this protein otherwise to have normal biochemical properties.

Ha-VP39 binding to actin and the influence of F-actin on assembly of progeny virions

We present evidence that actin is necessary for the successful assembly of HaNPV virions. Purified nucleocapsid protein Ha-VP39 of Heliothis armigera nuclear polyhedrosis virus (HaNPV) was found to be able to bind to actin in vitro without assistance, as demonstrated by Western blot and isothermal titration calorimeter. DeltaH and binding constants (K) detected by isothermal titration calorimeter strongly suggested that Ha-VP39 first binds actin to seed the formation of hexamer complex of actin, and the hexamers then link to each other to form filaments, and the filaments finally twist into cable structures. The proliferation of HaNPV was completely inhibited in Hz-AM1 cells cultivated in the medium containing 0.5 microg/ml cytochalasin D (CD) to prevent polymerization of actin, while its yield was reduced to 10(-4) in the presence of 0.1 microg/ml CD. Actin concentration and the viral DNA synthesis were not significantly affected by CD even though the progeny virions assembled in the CD treated cells were morphologically different from normal ones and resulted in fewer plaques in plaque assay.