Expression of lacZ reporter gene under the control of the polyhedrin promoter of Spodoptera litura nuclear polyhedrosis virus

Promoter function of the putative polyhedrin-encoding gene (polh) of Spodoptera litura nuclear polyhedrosis virus (S1MNPV) was determined by transferring it to the Autographa californica nuclear polyhedrosis virus (AcMNPV) through the AcNPV polh based vector, pVL1393. Three transfer vectors pCBT2, pCBT3 and pCBT4 were constructed by substituting the promoter and the neighbouring sequences of AcNPV in pVL1393 by that of SINPV. The Escherichia coli lacZ gene was placed downstream from the S1NPV polh promoter in the hybrid transfer vector (pCBT) constructs. Co-transfection of Spodoptera frugiperda cells (Sf9) with each of the pCBTlacZ vector and wild-type AcNPV DNAs led to synthesis of β-galactosidase (βGal). The plaque-purified recombinant viruses (S1AcNPV.lacZ) expressing lacZ under the polh promoter of S1NPV are stable. The highest βGal activity was obtained with S1AcNPV4.lacZ. Production of βGal with recombinant virus, S1AcNPV3.lacZ in which S1NPV polh promoter is in the reverse orientation in the AcNPV genome, is 83% of that produced by S1AcNPV4.lacZ. These results indicate that the S1NPV polh promoter is active in the genetic environment of AcNPV; the polh of S1NPV is phylogenetically related to AcNPV like other baculoviruses.     

Identification and characterization of the p35 gene of Bombyx mori nuclear polyhedrosis virus that prevents virus-induced apoptosis.

Nucleotide sequence analysis of the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome revealed the existence of a gene homologous to the p35 gene of Autographa californica NPV (AcNPV), which has been shown to prevent virus-induced apoptosis. The BmNPV p35 gene showed 96.1% nucleotide and 89.6% predicted amino acid sequence identity to the AcNPV p35 gene. A mutant BmNPV (BmP35Z) lacking a functional p35 gene induced apoptosis-like cell degradation in infected BmN cells. However, unlike the p35-deleted AcNPV mutant (vAcAnh), BmP35Z replicated normally and produced polyhedral inclusion bodies. The patterns of protein synthesis and the percentages of viable BmN cells remaining following infection with either wild-type BmNPV or BmP35Z were nearly identical. BmP35Z also replicated in silkworm larvae without showing any apparent apoptotic response in infected hemocytes, fat body, or other tissues. Time to death of larvae infected with BmP35Z was similar to that for wild-type-infected larvae, and significant numbers of polyhedral inclusion bodies were produced. These results indicate that viral factors (or genes) other than p35 or host cell factors play a role in inducing, accelerating, or interfering with apoptotic processes. The evolution of baculovirus genomes is also discussed with reference to comparative analysis of the p35 and p94 gene sequences. The p94 gene is found immediately upstream of p35 in AcNPV; in BmNPV, however, the p94 gene was nearly completely missing, presumably because of large deletions in a BmNPV ancestor virus having a gene similar to the AcNPV p94 gene.     

Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV,and knockout of cysteinase gene for more efficient expression

AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV(Bombyx mori nuclear polyhedrosis virus) are two principal insectbaculovirus expression systems, each having different characteristics. AcNPV has a wider host range and can infect a series of cell lines thus making it suitable for cell suspension culture expression, but the small size of the host insect,A. californica, makes AcNPV less suitable for large scale protein synthesis. In contrast, BmNPV can only infect the silkworm,Bombyx mori, which is wellknown for its easy rearing and large size. These characteristics make the BmNPV system especially suitable for largescale industrial expression. To utilize the advantages of both AcNPV and BmNPV, we tried to expand their host range through homologous recombination and successfully constructed a hybrid baculovirus of AcNPV and BmNPV, designated as HyNPV The hybrid baculovirus can infect the hosts of both AcNPV and BmNPV. Taking the human basic fibroblast growth factor (bFGF) gene as an application example, we constructed a recombinant, HyNPV-bFGF. This construct is able to express the bFGF protein both in silkworm larvae and in commonuse cell lines, sf21, sf9 and High-five. Moreover, to reduce the loss of recombinant protein due to degradation by proteases that are simultaneously expressed by the baculovirus, we knocked out the cysteinase gene coding for one of the most important baculovirus proteases. This knockout mutation improves the production efficiency of the bFGF recombinant protein.     

Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae

The full-length bovine interferon-gamma (bIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus transfer vectors pAcYM1 and pBm050. These vectors were co-transfected with Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA into Spodoptera frugiperda cells (SF21AE) and Bombyx mori cells (BmN), respectively. The recombinant viruses, named AcBIFN-gamma and BmBIFN-gamma, were then recovered. Recombinant bIFN-gamma (rbIFN-gamma) was accumulated in the culture fluid of AcBIFN-gamma-infected Trichoplusia ni cells and BmBIFN-gamma-infected silkworm larvae. These rbIFN-gamma forms were shown to be glycosylated 20 and 22 kDa proteins as confirmed by SDS-PAGE and tunicamycin treatment. These products were sensitive to cystein proteinase. Both rbIFN-gamma proteins, showed high-level biological activities by plaque reduction assay using vesicular stomatitis virus, and MHC class II antigen induction on bovine macrophage cells.     

Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV, and knockout of cysteinase gene for more efficient expression

The four main gene expression systems currently used to produce recombinant proteins are the prokary-otic, yeast, insect cell, and mammalian cell expression systems. The baculovirus expression vector system (BEVS) is a protein production system which uses a recombinant baculovirus harboring a foreign gene of interest to produce recombinant protein in an insect or its cultured cells. BEVS has many advantages: (i) BEVS requires less time to establish the production system than is needed in a…     

High level expression of a mutant (K151E, R154G) of single chain urokinase-type plasminogen activator in silkworm

A cDNA of a mutant (K151E, R154G) of single chain urokinase-type plasminogen activator (mscu-PA) was constructed to include the natural scu-PA signal peptide sequences and transferred into the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) by transfer vectors pBE284 (derived from BmNPV) and pVL1392 (from AcNPV), respectively. Both Bombyx mori (BmN) cells and silkworm larvae were infected with the two recombinant viruses. Fibrin-plate assay showed that the re-virus from pVL1392 increased the yield of mscu-PA three times compared with the re-virus from pBE284.     

Host range expansion by recombination of the baculoviruses Bombyx mori nuclear polyhedrosis virus and Autographa californica nuclear polyhedrosis virus.

The mechanisms of host specificity of nuclear polyhedrosis viruses (NPVs) (Baculoviridae) were analyzed after coinfection of Bombyx mori NPV (BmNPV) and one of four distinct groups of Spodoptera litura NPV (SlNPV), including an Autographa californica NPV (AcNPV) variant (S. Maeda, Y. Mukohara, and A. Kondo, J. Gen. Virol. 71:2631-2639, 1990), into various lepidopteran cell lines. Replication of BmNPV in nonpermissive cells (TN-386, SF-21, and CLS-79) was induced by coinfection with AcNPV but not with the other three SlNPV groups. These induced progeny NPVs were plaque purified in BmN cells, which are susceptible to only BmNPV, and characterized. Most of these isolates did not replicate in the cell lines in which they were produced, indicating the existence of a helper function of AcNPV for BmNPV replication in nonpermissive cells. Some of these isolates, however, were able to replicate in cell lines nonpermissive to BmNPV, indicating the appearance of a new virus with wider host specificity. DNA restriction endonuclease analysis showed that the isolates exhibiting wider host range were recombinant viruses between the parents, AcNPV and BmNPV, resulting from various types of crossovers of relatively large areas of their genomes. Expansion of host range was also observed in larvae.     

High-level expression and improved folding of proteins by using the vp39 late promoter enhanced with homologous DNA regions

Some recombinant proteins expressed by baculovirus expression vector systems (BEVS) aggregate because the BEVS can produce large amounts of protein late during infection, when post-translational modification and protein quality control mechanisms are inactive. For expression during earlier stages than that driven by the polyhedrin (polh) very late promoter, transfer vectors were generated in which this promoter was replaced with a green fluorescent protein (GFP) gene controlled by a vp39 late promoter modified to contain HR3, one of the homologous DNA regions (HRs) of Bombyx mori nuclear polyhedrosis virus (BmNPV). The rise times of the fluorescence of GFP expressed by using recombinant viruses carrying the modified vp39 promoter were earlier than those associated with either the polh promoter or the native vp39 promoter lacking HR3. In transient expression assays, the vp39 late promoter in transfer vectors behaved like a delayed-early promoter, and was enhanced by HR3, and required IE-1 protein and various viral gene products encoded on both sides of BmNPV polh. When the vp39 promoter with HR3 was used, the aggregation of several foreign proteins expressed by the BEVS was markedly decreased. This study provides a new option for the expression of sufficiently quality-controlled proteins by using the vp39 promoter and HR3 in BEVS early in baculovirus infection, when the infection has caused little damage in the host cells.     

Host range expansion of Autographa californica nuclear polyhedrosis virus (NPV) following recombination of a 0.6-kilobase-pair DNA fragment originating from Bombyx mori NPV.

We have isolated hybrid baculoviruses of Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica NPV (AcNPV) capable of replicating in both BmN (not susceptible to AcNPV) and SF-21 (not susceptible to BmNPV) cells (A. Kondo and S. Maeda, J. Virol. 65:3625-3632, 1991). Repeated backcross infection of one of these recombinant isolates with AcNPV generated eh-AcNPV, a virus with restriction endonuclease patterns of genomic DNA nearly identical to those of AcNPV but capable of replicating in both BmN and SF-21 cells, i.e., host range expanded. Expanded host range viruses were also isolated following cotransfection of AcNPV DNA with eh-AcNPV DNA cleaved with either HindIII or PstI. Subsequent cotransfection of AcNPV DNA with plasmids from an eh-AcNPV DNA fragment library identified an 11-kbp HindIII fragment that could expand the host range of AcNPV. Subcloning and cotransfection analyses localized a 572-bp SacI-HindIII fragment within this 11-kbp fragment which could alone expand the host range of AcNPV. Mapping and nucleotide sequencing analysis revealed that this fragment was identical to the corresponding 572-bp fragment (BmScH) of BmNPV. Furthermore, this fragment originated from the coding region of the putative DNA helicase gene. Cotransfection of AcNPV DNA with BmScH also generated a host range-expanded virus, eh2-AcNPV. These results indicated that the expanded host range characteristics of eh2-AcNPV were solely the result of recombination within the coding region of the putative DNA helicase gene.     

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system

Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.     

Divergence and evolution of homologous regions of Bombyx mori nuclear polyhedrosis virus.

Homologous regions (hrs) (hr1,hr2-left,hr2-right,hr3,hr4-left,hr 4-right, and hr5) similar to those found in the Autographa californica nuclear polyhedrosis virus (AcNPV) genome were found in the Bombyx mori NPV (BmNPV) genome. The BmNPV hrs contained two to eight repeats of a homologous nucleotide sequence which were on average about 75 bp long. All of these homologous sequence repeats contained a 26-bp-long palindrome motif with an EcoRI or EcoRI-like site at its core. The consensus sequence of the BmNPV hrs showed 95% conservation with respect to those found in AcNPV. Nucleotide sequence analysis indicated that hr2-left and hr2-right of BmNPV evolved from an ancestor similar to hr2 of AcNPV by inversion, cleavage, and ligation. The polarities of the BmNPV and AcNPV hrs were conserved except for that of hr4-left. Within hr4-right of BmNPV, four repeats of a previously underscribed palindrome motif were found. Bmhr5D, a BmNPV mutant which lacked hr5, replicated at a rate similar to that of wild-type BmNPV in BmN cells and silkworm larvae, indicating that hr5 was not essential for viral replication. After ten passages of Bmhr5D in BmN cells, no detectable changes in its genome were observed by restriction endonuclease analysis. The evolution and divergence of the BmNPV genome are also discussed.     

Expression of a foreign gene by cysteine proteinase null recombinant baculovirus

Baculovirus expression vector systems (BEVSs) are broadly used for producing foreign proteins in lepidopteran cells. Most commercial BEVSs are engineered to insert foreign genes into the polyhedrin (polh) locus. They lack the polh gene. These viruses cannot produce occlusion bodies and are inconvenient for per os inoculation of larvae. To avoid this, expression cassettes can be inserted in other parts of the virus genome. The preS2-S gene, coding for the recombinant middle surface antigen of the human hepatitis B virus (M-HBsAg), was expressed from the baculovirus construct rBmNPV-Δv-cath-M-HBsAg, inserting the foreign gene into the v-cath locus of Bombyx mori nucleopolyhedrovirus (BmNPV) so that v-cath was deleted and native polh was retained. Silkworm larvae were infected per os and M-HBsAg was observed to be abundantly produced till very late stages of infection. Infection of larvae with a mixture of the recombinant and wild-type baculoviruses was followed by degradation of the bulk of the produced M-HBsAg as early as 96 h after inoculation.     

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.     

Development of baculovirus triple and quadruple expression vectors: co-expression of three or four bluetongue virus proteins and the synthesis of bluetongue virus-like particles in insect cells.

Baculovirus multiple gene transfer vectors pAcAB3 and pAcAB4 have been developed to facilitate the insertion of three or four foreign genes respectively into the Autographa californica nuclear polyhedrosis virus (AcNPV) genome by a single co-transfection experiment. The pAcAB3 vector contains a polyhedrin promoter and two p10 promoters on either side of the polyhedrin promoter but in opposite orientations. The pAcAB4 vector has an additional polyhedrin promoter in opposite orientation to the first copy that is in juxtaposition to the first p10 promoter. Each of these derived vectors (pAcAB3, pAcAB4) have been used for the simultaneous expression of three or four bluetongue virus (BTV) genes respectively. When Spodoptera frugiperda cells were infected with the recombinant virus (AcBT-3/2/7/5) expressing the four major structural genes of BTV, double-capsid, virus-like particles consisting of VP2, VP3, VP5 and VP7 of BTV were assembled.     

以核多角体病毒为载体在家蚕中生产外源蛋白

以家蚕核多角体病毒(BmNPV)为载体,在家蚕幼虫或家蚕培养细胞系中表达的外源基因越来越多,其表达的产物已涉及到医用药物、医疗诊断、疫苗生产、生物防治等诸多领域,文章就BmNPV的特性及其基因组构造,多角体蛋白基因的特性,重组BmNPV的构建及其在家蚕幼虫体内和细胞系中的表达,BmNPV-家蚕表达系统的外源蛋白生产效率及其应用等各个方面作了全面、系统的综述.     

Sequencing of the putative DNA helicase-encoding gene of the Bombyx mori nuclear polyhedrosis virus and fine-mapping of a region involved in host range expansion

The complete sequence of a 3666-nucleotide (nt) open reading frame (ORF) and its flanking regions (58.1–62.1 map units (m.u.) from Bombyx mori nuclear polyhedrosis virus (BmNPV)) was determined. This ORF, BmNPV dnahel, encoded a predicted protein of 143 623 Da which possessed seven consensus motifs found in proteins which unwind duplex DNAs, indicating that it is a DNA helicase. A 572-bp SacI-HindlII fragment, BmScH, that was previously shown to expand the host range of Autographa californica NPV (AcNPV) following homologous recombination [Maeda et al. (1993) J. Virol. 67, 6234–6238], was localized within BmNPV dnahel. By cotransfection experiments, two adjacent nt (A and T) that appeared to be the minimal essential sequence necessary to expand the host range of AcNPV, were mapped within BmScH. These adjacent nt encoded a single amino acid difference between BmNPV (Asp) and AcNPV (Ser).