Isolation of p10 gene fromBombyx mori nuclear polyhedrosis virus and study of its promoter activity in recombinant baculovirus vector system

A homologue ofAutographa californica NPV (AcNPV) p10 gene was identified and cloned fromBombyx mori NPV (BmNPV). BmNPV p10 gene encodes truncated protein of 70 amino acid residues that lacks carboxyl terminus comparing with the p10 protein encoded by AcNPV. The putative TATA box sequence and the ATAAG motif which is the consensus sequence of baculovirus very late promoter were conserved. A transfer vector, pBNT1, which includes the p10 promoter region of BmNPV for foreign gene expression was constructed. By using pBNT1, a recombinant BmNPV, Bmp10-Luc, in which the p10 gene was replaced by the firefly luciferase gene, was obtained. We also obtained another recombinant virus, BmPH-Luc, in which the polyhedrin gene was replaced by the luciferase gene. The luciferase activity detected in BoMo-15AIIc insect cells infected with Bmp10-Luc was approximately 50% of that infected with BmPH-Luc, suggesting that although both the p10 and polyhedrin promoters of BrnNPV are effective in high-level expression of foreign gene, the p10 promoter is not so strong as the polyhedrin promoter.     

Investigation of sequential behavior of carboxyl protease and cysteine protease activities in virus-infected Sf-9 insect cell culture by inhibition assay

Proteases produced during the culture of Spodoptera frugiperda Sf-9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV) were assayed with various protease inhibitors. This inhibitory analysis revealed that: (1) carboxyl and cysteine proteases were predominantly produced by the insect cells infected with recombinant AcNPV, the gene of which encoded a variant of green fluorescent protein in a portion of the polyhedrin gene of the baculovirus, and (2) the protease activity was almost completely blocked by pepstatin A (carboxyl protease inhibitor) and E64 (cysteine protease inhibitor) in an additive manner in the presence of EDTA. Utilizing the additive property of the inhibitors, the inhibition-based protease assay discriminated between the two protease activities and elucidated the sequential behavior of the carboxyl and cysteine proteases produced in the virus-infected Sf-9 cell culture. The carboxyl protease(s) existed in the virus-infected cells all the time and their level in the medium continuously increased. Uninfected cells also contained a carboxyl protease activity, the level of which was similar to that of the virus-infected cells. At a certain time after virus infection, the cysteine protease activity was largely increased in the virus-infected cells and a significant amount of the protease(s) was released into the medium, due to the cell membranes losing their integrity. The behavior of intracellular and extracellular cysteine protease activities coincided with that of a recombinant protein whose expression was under the control of the viral polyhedrin promoter. Similar examinations with wt-AcNPV-infected and uninfected insect cells showed that the inhibition-based protease assay was useful for analyzing the carboxyl protease and cysteine protease activities emerging in the insect cell (Sf-9)/baculovirus expression system.     

Bruton酪氨酸激酶在昆虫细胞中表达的初步研究

用杆状病毒表达载体系统表达小鼠Ｂｒｕｔｏｎ酪氨酸激酶（Ｂｒｕｔｏｎｔｙｒｏｓｉｎｅｋｉｎａｓｅ,Ｂｔｋ）．构建重组转染载体时,于Ｂｔｋ起始码的上游插入了一段Ｈ９０２序列．用重组转染载体、苜蓿夜蛾核型多角体病毒线性ＤＮＡ和质脂体共转染Ｓｆ９昆虫细胞,经过对重组杆状病毒三轮扩增后,用Ｈ９０２抗体检测表明,昆虫细胞中Ｂｔｋ的表达已达最高水平．对表达后Ｂｔｋ自身磷酸化检测表明,该激酶具有自身磷酸化活性．从而证实Ｂｔｋ在昆虫细胞中的表达获得了成功     

Insecticidal Activity of a Bacterial Crystal Protein Expressed by a Recombinant Baculovirus in Insect Cells

Baculoviruses are insect pathogens with a relatively slow speed of action, and this has limited their use as control agents of insect pests. Introduction into baculoviruses of genes which code for proteins interfering specifically with insect metabolism or metamorphosis, such as toxins, hormones, and enzymes, may enhance the pathogenicity of these viruses. The complete insecticidal crystal protein gene cryIA(b) of Bacillus thuringiensis subsp. aizawai 7.21 was engineered into the nuclear polyhedrosis virus of Autographa californica (AcNPV) in place of the polyhedrin gene. In infected Spodoptera frugiperda cells, the cryIA(b) gene was expressed at a high level without interference with AcNPV production. The crystal protein was found in the cytoplasm of S. frugiperda cells, mainly as large crystals with an ultrastructure similar to that of B. thuringiensis crystals. Infected-cell extracts inhibited feeding of the large cabbage white Pieris brassicae. The toxicity of the crystal protein expressed by AcNPV recombinants was comparable with that of the crystal protein expressed by a corresponding Escherichia coli recombinant.     

Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector.

The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has played a major role in studies on the molecular biology of insect DNA viruses. Recently, this system has been effectively adapted as a highly efficient vector in insect cells for the expression of several mammalian genes. A cDNA sequence of the influenza (fowl plague) virus haemagglutinin gene has been inserted into the BamHI site of the pAc373 polyhedrin vector. Spodoptera frugiperda cells were co-transfected with this construct, pAc-HA651, and authentic AcNPV DNA. Recombinant virus was selected by adsorption of transfected cells to erythrocytes followed by serial plaque passages on S. frugiperda cells. We have determined the site of insertion of the haemagglutinin gene into the AcNPV genome by restriction enzyme cleavage and Southern blot hybridization analyses using haemagglutinin cDNA as a probe. The influenza haemagglutinin gene is located in the polyhedrin gene of AcNPV DNA. Immunofluorescent labelling, immunoprecipitation and immunoblot analyses with specific antisera revealed that S. frugiperda cells produce immune reactive haemagglutinin after infection with the recombinant virus. The haemagglutinin is expressed at the cell surface and has haemolytic capacity that has been activated by post-translational proteolytic cleavage. When chickens were immunized with S. frugiperda cells expressing haemagglutinin, they developed haemagglutinin-inhibiting and neutralizing antibodies and were protected from infection with fowl plague virus. These observations demonstrate that the haemagglutinin is processed in insect cells in a similar fashion as in fowl plaque virus-infected vertebrate cells and that it has full biological activity.     

Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector.

The N-terminal region of the gene encoding polyhedrin, the major occlusion protein of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), has been fused to DNA encoding Escherichia coli beta-galactosidase. The fused gene was inserted into the AcNPV DNA genome by cotransfection of insect cells with recombinant plasmid DNA and wild-type AcNPV genomic DNA. Recombinant viruses were selected as blue plaques in the presence of a beta-galactosidase indicator, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Studies of one such virus, L1GP-gal3, indicated that the synthesis of beta-galactosidase is temporally controlled beginning late (20 h) in infection after the release of infectious virus particles from the cell. By 48 h postinfection, a remarkably high level of expression is achieved. On the basis of these results, AcNPV should be a useful vector for the stable propagation and expression of passenger genes in a lepidopteran cell background. A generalized transplacement vector that facilitates the construction and selection of recombinant viruses carrying passenger genes under their own promoter control has also been developed.     

Expression of human tyrosine hydroxylase cDNA in invertebrate cells using a baculovirus vector

A human cDNA containing the complete coding sequence for a human tyrosine hydroxylase (EC 1.14.16.2, form 2) was introduced into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) downstream to the polyhedrin promoter. Infection of Spodoptera frugiperda cells (SF9) with recombinant virus resulted in the expression of human tyrosine hydroxylase in these invertebrate cells. Characterization of tyrosine hydroxylase activity in infected SF9 cells demonstrated both substrate and cofactor kinetics that were characteristic of those previously reported for the native human enzyme. Both 3-iodotyrosine and alpha-methyl-p-tyrosine competitively inhibited the recombinantly produced tyrosine hydroxylase with Ki values of 1.2 and 16 microM, respectively, similar to those previously reported for the rat and human enzymes. Western blot analysis of extracts of SF9 cells infected with the recombinant baculovirus containing human tyrosine hydroxylase cDNA revealed a major immunoreactive band with an apparent Mr of 60 kDa, identical to the size of the immunoreactive protein from rat adrenal and caudate nucleus. The use of the baculovirus expression system to produce abundant quantities of each of the multiple forms of active human tyrosine hydroxylase in eukaryotic cells should facilitate structural analysis and help clarify the physiological significance of each of the isoenzymes.     

Expression and characterization of the chick nicotinic acetylcholine receptor alpha-subunit in insect cells using a baculovirus vector

A baculovirus transfer vector was constructed containing an entire cDNA copy of the chick nicotinic acetylcholine receptor (nAChR) alpha-subunit under control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene promoter. Recombinant baculovirus was obtained by co-transfection of Spodoptera frugiperda cells with infectious, wild-type AcNPV DNA and the transfer vector. Polyhedrin-negative, recombinant viruses were identified which expressed the nAChR alpha-subunit. The insect cell-expressed alpha-subunit protein had a molecular mass of 42 kDa and was shown to be targeted to the plasma membrane by fluorescence microscopy and toxin-binding assays. The levels of expression were low, approximately 1-2% of cell proteins, when compared with the levels of natural polyhedrin protein. The expressed receptor alpha-subunit was recognised by polyclonal antisera raised against purified Torpedo nAChR alpha-subunit and carried the binding site for the snake venom toxin, alpha-bungarotoxin. Bound alpha-bungarotoxin was displaced in competition binding assays by alpha-cobra toxin, carbamylcholine and d-tubocurarine, and thus had a similar pharmacological profile to that obtained with authentic receptors in muscle cells and receptors expressed in other systems i.e. Xenopus oocytes and mammalian cells. We have also shown that when the chick nAChR alpha-subunit is expressed in the absence of other receptor subunits, unexpectedly high concentrations of nicotine (10 mM) were required to displace bound alpha-bungarotoxin.     

Hepatocyte-specific gene expression by baculovirus pseudotyped with vesicular stomatitis virus envelope glycoprotein.

We have developed the recombinant baculovirus pseudotyped with vesicular stomatitis virus (VSV) G protein. The VSV-G gene was under the control of the polyhedrin promoter so that it was expressed at high levels in infected insect cells but not in mammalian cells. The presence of VSV-G protein in purified baculovirus preparations was confirmed by Western analysis. This recombinant baculovirus also carried human AFP (alpha-fetoprotein) promoter for hepatocyte-specific gene expression. After an in vitro infection by a recombinant baculovirus carrying the luciferase gene under the control of human AFP promoter/enhancer (BacG-AFP-Luc(+)), the luciferase gene was expressed in AFP-producing Huh7, Hep3B, and HepG2 cell lines, but not in AFP-nonproducing cell lines. BacG-AFP-Luc(+) transduced with human hepatoma cells in vitro at an efficiency about fivefold greater than the recombinant baculovirus lacking VSV-G (the virus Bac-AFP-Luc(+)). The utilization of the AFP promoter/enhancer in a baculovirus vector could provide benefits in gene therapy applications.     

Identification of bluetongue virus VP6 protein as a nucleic acid-binding protein and the localization of VP6 in virus-infected vertebrate cells.

Recently the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been effectively adapted as a highly efficient vector in insect cells for the expression of various genes. A cDNA sequence of RNA segment 9 of bluetongue virus serotype 10 (BTV-10, an orbivirus member of the Reoviridae family) encoding a minor core protein (VP6) has been inserted into the BamHI site of the pAcYM1 transfer vector derived from AcNPV. Spodoptera frugiperda cells were cotransfected with the derived vector in the presence of authentic AcNPV DNA to produce recombinant viruses. These synthesized significant amounts of a protein (representing ca. 50% of the stained cellular protein) similar in size and antigenicity to the authentic BTV VP6. The expressed protein was identified as a nucleic acid-binding protein by using an RNA overlay-protein blot assay. A polyclonal anti-VP6 serum prepared by using the expressed VP6 protein has been used in an immunogold procedure to locate VP6 in BTV-infected mammalian cells. Gold was found to be associated with the matrix of virus inclusion bodies (VIB), with viruslike particles in the VIB, as well as with mature virion particles that were in close proximity to the VIB or were released from cells and adsorbed to cell surfaces. The recombinant virus antigen has also been used to identify antibodies to different BTV serotypes in infected sheep sera, indicating the potential of the expressed protein as a group-reactive antigen for the diagnosis of BTV infections.     

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.     

Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.     

绿色荧光蛋白基因在昆虫细胞中的克隆与表达

将绿色荧光蛋白(GFP)基因亚克隆到转移载体pVLneo的多角体蛋白基因(ocu)启动子下游,与杆状病素AcNPV DNA共转染昆虫细胞,通过同源重组和G418筛选,构建了整合有GFP基因的重组病毒。在昆虫细胞中表达的GFP,MW为30kDa,在荧光显微镜下呈现美丽的绿色,荧光光谱表明其激发波长395nm,发射波长509nm。Southern blot杂交证明,重组病毒的1kb EcoRI片段与GFP cDNA探针有很强的杂交信号,这是GFP基因在杆状病毒基因组中整合的直接证据。     

Baculovirus expression system for magnetic sorting of infected cells and enhanced titer determination

Recombinant baculoviruses derived from the Autographa californica nuclear polyhedrosis virus (AcNPV) are widely used to express heterologous genes in insect cells, but the use of the baculovirus expression vector system (BEVS) is hampered by slow and tedious procedures for the selection and separation of baculovirus-infected insect cells and for titer determination. Here we developed a new technology based on the bicistronic vector with a fusion protein of the human integral plasma membrane glycoprotein CD4 and green fluorescent protein (GFP) for concomitant expression of target proteins in insect Sf21 cells. Magnetic cell sorting (MACS) technology with anti-CD4 antibody-labeled superparamagnetic beads was used to separate the baculovirus-infected from the noninfected insect cells and therefore to increase the virus titer and to reduce process time. With the herein described use of the MACS-improved baculovirus expression plasmid MACS in baculovirus expression (pMACSiBac-1), we have been able to select the baculovirus-infected insect cells at an early time point of the infection cycle and therefore enrich the virus titer dramatically. Furthermore, simple end point dilution and GFP fluorescence detection can be used for early and facile detection of recombinant viruses and simplified titer determinations. We show that the bicistronic pMACSiBac-1 with an additional multiple cloning site under the control of the very late promoter polyhedrin (PPH) allows for the expression of target proteins in high amounts, less workloads, and shorter timelines.     

Development of an antibody-based assay for determination of baculovirus titers in 10 hours

The baculovirus expression system has been used to produce large amounts of biologically active proteins by infecting insect cells with a recombinant baculovirus expressing the target protein. For an efficient expression of the target protein, it is necessary to infect insect cells with an adequate amount of virus. However, current methods are time-consuming and either have technical difficulties or are limited as a result of virus expression mechanism using a reporter gene. A novel method is developed to yield virus titers in 10 h that is easy to perform using 96-well plates and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immunostaining. The titer is determined by counting foci produced as a result of infection of the virus under a fluorescent microscope. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post-infection time of 4 h. Therefore, 10 h was enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer. Titers determined using this immunological assay are comparable, both in value and validity, to those obtained using a traditional method, provided that the stocks have titers above 10(3) pfu/mL.     

Expression of human immunodeficiency virus genes using baculovirus expression system

The structural protein genes of HIV-1 and HIV-2 have been expressed inSpodoptera frugiperda (SF) cells using baculovirus expression system. The noncoding flanking sequences of HIV structural genes were removed and a putative ribosome binding site was placed in front of the open reading frame of each gene by using crossover linker mutagenesis. The coding sequences of thegag, pol, env, andvif proteins were inserted intoAutographa californica nuclear polyhedrosis virus (AcNPV) so that HIV genes were under the control of the AcNPV polyhedrin promoter. All recombinant AcNPV-infected SF cells express high levels of HIV structural proteins. Detailed strategies of recombinant AcNPV construction for high level protein expression are presented.