Functional analysis and molecular characterization of two acetylcholinesterases from the German cockroach, Blattella germanica.

Two acetylcholinesterases (AChEs; BgAChE1 and BgAChE2) from Blattella germanica were functionally expressed using the baculovirus system. Kinetic analysis demonstrated that BgAChE2 had higher catalytic efficiency but lower substrate specificity than BgAChE1. With the exceptions of paraoxon and propoxur, BgAChE1 was generally less sensitive to inhibitors than BgAChE2. Western blot analysis using anti-BgAChE antibodies revealed that BgAChE1 was far more abundant in all examined tissues compared to BgAChE2, which is only present in the central nervous system. Both BgAChEs existed in dimeric form, covalently connected via a disulphide bridge under native conditions. Most fractions of BgAChE1 had a glycophosphatidylinositol (GPI) anchor, but a small fraction comprised a collagen-like tail. BgAChE2 appeared to have a collagen-GPI-fused tail. Based on the kinetic and molecular properties, tissue distribution and abundance, BgAChE1 was confirmed to play a major role in postsynaptic transmission.

Suppression of tumor growth in xenograft model mice by programmed cell death 4 gene delivery using folate-PEG-baculovirus.

Cancer gene therapy using tumor suppressor genes is considered to be an attractive approach for arresting cell growth and inducing apoptosis. Programmed cell death 4 (Pdcd4) is a tumor suppressor gene, which prevents tumorigenesis and tumor progression. To address the issue of whether expression of PDCD4 protein induces apoptosis in cancerous cells, the Pdcd4 gene was delivered using folate-PEG-baculovirus. Folate-PEG-baculovirus containing Pdcd4 gene (F-P-Bac-Pdcd4) was constructed by attachment of F-PEG to the baculovirus surface using chemical modification. The F-P-Bac-Pdcd4 showed enhanced transduction efficiency, efficiently expressed PDCD4 protein, and induced apoptosis in human epidermal carcinoma (KB) cells as compared with an unmodified baculovirus. In a tumor xenograft study, injection of F-P-Bac-Pdcd4 into tumors established from the KB cell line by subcutaneous implantation significantly suppressed tumor growth and induced apoptosis. Thus, this study shows a new baculovirus-mediated tumor suppressor gene delivery system for cancer therapy.

Expression and characterization of a recombinant Cry1Ac crystal protein with enhanced green fluorescent protein in acrystalliferous Bacillus thuringiensis.

AIMS: To investigate fusion expression between Bacillus thuringiensis crystal protein and a foreign protein, the expression of a fusion protein comprised of Cry1Ac, and enhanced green fluorescent protein (EGFP) in B. thuringiensis Cry(-)B strain was examined. METHODS AND RESULTS: The N-terminal fusion expression of EGFP in Cry1Ac was attempted under the control of the native cry1Ac promoter. The EGFP gene was cloned into pProMu and named pProMu-EGFP. The transformant, ProMu-EGFP/CB produced parasporal inclusions that were of bipyramidal-shaped crystals in size ranging from 200 to 300 nm. The fusion protein was approximately 150 kDa and identified by the immunoblot analysis using a Cry1Ac antibody and also a GFP antibody. The LC(50) of the ProMu-EGFP/CB was twofold higher when compared with that by the ProAc/CB. However, the crystal protein produced by the ProMu-EGFP/CB was effective on Plutella xylostella larvae. CONCLUSIONS: The ProMu-EGFP/CB produced bipyramidal shaped and insecticidal crystals comprising fusion proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the N-terminal fusion expression of EGFP and Cry1Ac, expression and crystallization between the B. thuringiensis crystal protein and a foreign protein were validated.

Baculovirus expression vectors that incorporate the foreign protein into viral occlusion bodies.

Current baculovirus expression systems typically produce soluble proteins that accumulate within the infected insect cell or are secreted into the growth medium. A system has now been developed for the incorporation of foreign proteins, along with the matrix protein, polyhedrin, into baculovirus occlusion bodies. Initial studies showed that a recombinant virus expressing a translational fusion between polyhedrin and GFP did not form occlusion bodies. However, a baculovirus coexpressing native polyhedrin and the polyhedrin-GFP fusion protein formed occlusion bodies that fluoresced under UV light, demonstrating that they included the polyhedrin-GFP fusion protein. This was confirmed by immunoblot analysis. Thus, incorporation of a foreign protein into occlusion bodies depends on an interaction between native polyhedrin and the polyhedrin fusion protein. Electron microscopy demonstrated that the occlusion bodies containing GFP also incorporated virions as expected. These ColorPol occlusion bodies were as infectious to insect larvae as occlusion bodies produced by wild-type virus. This new system expands the capabilities for foreign gene expression by baculoviruses, which has implications for biopesticide design, novel vaccine delivery systems, and fusion protein purification applications.

Isolation and characterization of a strain of Bacillus thuringiensis ssp. kurstaki containing a new delta-endotoxin gene.

A strain of Bacillus thuringiensis that showed significantly high toxicity to Plutella xylostella and Spodoptera exigua was isolated from a Korean soil sample and characterized. The isolate, named B. thuringiensis K1, was determined to belong to ssp. kurstaki (H3a3b3c) type by an H antisera agglutination test and produced bipyramidal inclusions. Plasmid pattern of K1 was different from that of the reference strain, ssp. kurstaki HD-1, but the parasporal inclusion protein profile of K1 had two major bands that were similar in size to those of ssp. kurstaki HD-1. To verify the delta-endotoxin gene types of K1, PCR analysis with specific cry gene primers was performed to show that K1 contained a new cry gene in addition to cry1Aa, cry1Ab, cry1Ac, cry1E and cry2 genes. PCR-amplified region of the new cry gene, cryX, showed 79% similarity to cry1Fa1 gene (GenBank Accession No. M63897). In an insect toxicity assay, K1 had higher toxicity against Plutella xylostella and S. exigua than ssp. kurstaki HD-1.

Isolation and characterization of a Bacillus thuringiensis ssp. kurstaki strain toxic to Spodoptera exigua and Culex pipiens.

A strain of Bacillus thuringiensis with dual toxicity was isolated from Korean soil samples and named K2. K2 was determined as ssp. kurstaki (H3a3b3c) by serological test and produced bipyramidal-shaped parasporal inclusions. The plasmid and protein profiles of B. thuringiensis K2 were different from those of the reference strain, ssp. kurstaki HD-1. To verify gene type of B. thuringiensis K2, PCR analysis with specific cry gene primers was performed. The result showed that B. thuringiensis K2 had cry1Aa, cry1Ab, cry1C, and cry1D type genes, whereas ssp. kurstaki HD-1 had cry1Aa, cry1Ab, cry1Ac, and cry2 type genes. In addition, B. thuringiensis K2 had high toxicity against Spodoptera exigua and Culex pipiens, whereas B. thuringiensis ssp. kurstaki HD-1 does not have high toxicity against these two insect species.

High-level expression of canine parvovirus VP2 using Bombyx mori nucleopolyhedrovirus vector.

For the potential use as recombinant vaccine, canine parvovirus (CPV) major capsid protein VP2 was expressed using Bombyx mori nucleopolyhedrovirus (BmNPV) vector. CPV VP2 gene was introduced into polyhedrin-based BmNPV transfer vector pBmKSK3, and recombinant virus BmK1-Parvo was prepared. When anti-CPV.VP2 monoclonal antibody was employed in immunofluorescence staining, an intense signal was observed within BmK1-Parvo-infected Bm5 cells but not within uninfected cells or cells infected with a wild-type BmNPV-K1. In hemagglutination assay, the expression level of VP2 were 3.2 x 10(3) HA units/ml from infected Bm5 cells, 2.1x 10(5) HA units/larvae from infected larval fat body, and 1.6x 10(6) HA units/ml from infected larval hemolymph. These results suggested that BmNPV vector system using B. mori larva as host could be applied to efficient mass-production of recombinant vaccines.

Characterization of four isolates of Bombyx mori nucleopolyhedrovirus.

Bombyx mori nucleopolyhedroviruses (BmNPVs), isolated from a sericultural Korean farm, were purified and characterized by their DNA restriction pattern, virus replication, polyhedra production and gene structures. The EcoR I and Sal I fragments showed similar overall patterns with minor difference but distinguishable patterns in each isolate. There was no significant difference in the virus replication pattern, yield of total polyhedra production and polyhedra morphology, but the yield of released polyhedra by BmNPV-K1 in Bm5 cells was 2 to 5 times higher than that of other isolates. In comparative studies of p10 gene, BmNPV-K1 and K3 had same structure and they encoded a protein consisting of 94 amino acids. Although BmNPV-K2 encoded the same length of amino acids with BmNPV-K1 and K3, it had different structure, and BmNPV-K4 had the p10 gene encoding 70 amino acids.

Development of a novel expression vector system using Spodoptera exigua nucleopolyhedrovirus.

To develop a novel Spodoptera exigua nucleopolyhedrovirus (SeNPV) expression vector system, we examined characteristics of the SeNPV polyhedrin expression in S. exigua cells (Se301). While the extracellular virus titer of SeNPV was 100-fold lower than that of Autographa californica nucleopolyhedrovirus (AcNPV), the levels of polyhedral inclusion body (PIB) formation and polyhedrin expression were higher in SeNPV. To investigate foreign gene expression under the control of the polyhedrin promoter, polyhedrin-based transfer vector pSeKSK2 was constructed, and then recombinant virus SeK1-LacZ was constructed by inserting E. coli lacZ gene as a reporter gene into a genomic DNA of SeNPV using this transfer vector. The beta-Galactosidase activity of SeK1-LacZ in Se301 was about 1.3 times higher than that of BacPAK6. Thus, the SeNPV expression vector system constructed in this study would be very useful in the expression of foreign proteins, specifically for the enhancement of the pesticidal properties of SeNPV by inserting pesticidal genes.

Isolation of a strain of Bacillus thuringiensis ssp. kurstaki HD-1 encoding delta-endotoxin Cry1E.

A strain of Bacillus thuringiensis, STB-1, toxic against Spodoptera exigua, was isolated. Bacillus thuringiensis STB-1 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis ssp. kurstaki. The plasmid and protein profiles of B. thuringiensis STB-1 were compared with those of its reference strains, ssp. kurstaki and ssp. kenyae. To verify the gene type of B. thuringiensis STB-1, PCR analysis was performed with Spodoptera-specific cry gene primers. The result showed that B. thuringiensis STB-1, unlike its reference strains, had crylAa, crylAb, crylAc and crylE, suggesting that B. thuringiensis STB-1 was a unique strain with respect to gene type. In addition, B. thuringiensis STB-1 showed a high level of toxicity against both S. exigua and Bombyx mori, whereas B. thuringiensis ssp. kurstaki HD-1 or ssp. kenyae showed a high level of toxicity against only Bombyx mori or S. exigua, respectively.

Cloning and expression of a novel gene encoding a new antibacterial peptide from silkworm, Bombyx mori.

We differentially screened a novel gene encoding a new antibacterial peptide from the immunized Bombyx mori cDNA library. The gene showed a similar structure to that of cecropin-family, encoding 59 amino acids including a putative leader peptide and mature peptide. The deduced peptide, named Enbocin, had conserved amino acid residues which have been known to play an important role in the antibacterial activities. Enbocin genomic sequence revealed that the transcription unit of Enbocin gene was about 1.2 kb, and the coding sequence was interrupted by an intron of 660 bases. Recombinant Enbocin, expressed under the control of the baculovirus polyhedrin promoter, demonstrated a broad range of antibacterial activities against gram positive and gram negative bacteria.