Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order + 35 > + 1 > − 3 > − 8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60–72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in ‘glowing silkworms’. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.     

Efficient Protein Expression in <Emphasis Type="Italic">Bombyx mori</Emphasis> Larvae of the Strain d17 Highly Sensitive to <Emphasis Type="Italic">B. mori</Emphasis> Nucleopolyhedrovirus

The recombinant protein expression by Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworm larvae or pupae may endow us with a potent system for the production of large eukaryotic proteins. However, the screening of silkworm strains ideally suited to this method has scarcely been conducted. In the present study, we injected recombinant BmNPV containing a reporter gene, luciferase or DsRed, into hemocoel of fifth instar larvae of selected 12 silkworm strains. Among them, the strain d17 is found to be the highest in reporter expression from the intrinsic polyhedrin promoter of Autographa californica NPV or the silkworm actin A3 promoter. These results suggest that the d17 strain is highly permissive for BmNPV replication and is the most likely candidate of a “factory” for large-scale expression using the BmNPV bacmid system.     

Expression of EGFP driven by BmNPV orf4 promoter, a novel immediate early promoter

Bombyx mori nucleopolyhedrovirus (BmNPV) orf4 has been shown to be expressed at very early stage of Bm-NPV infection cycle. In this study, using transient expression experiment, we demonstrated for the first time that orf4 promoter is an immediate early promoter, indicating that orf4 may play a role in the immediate-early stage of BmNPV infection. Moreover, with the recently developed Bac-to-Bac/BmNPV baculovirus expression system and a modified pFast-Bac1 whose polyhedrin promoter was replaced with orf4 promoter, a recombinant bacmid baculovirus expressing enhanced green fluorescent protein (EGFP) under the control of orf4 promoter in Bombyx mori (Bm) cells was successfully constructed. The result not only showed that the polyhedrin promoter can be replaced easily with other promoters to direct the expression of foreign genes by using this novel system but also laid the foundation for the rescue experiment of orf4 deletion mutant.     

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.     

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.     

Bombyx mori nuclear polyhedrosis virus-based vectors for expressing passenger genes in silkmoth cells under viral or cellular promoter control

The polyhedrin gene of the nuclear polyhedrosis virus of the silkmoth Bombyx mori (BmNPV) has been subjected to deletion mutagenesis. A number of clones containing partially deleted polyhedrin genes were characterized and four clones containing limited deletions of the 5'-untranslated or 5'-flanking sequences of the gene were further analyzed with respect to polyhedrin promoter activity. The functional characterization of the deletion mutants was achieved through the insertion of a chloramphenicol acetyl transferase (CAT) gene (cat) into each deletion junction. The resultant cat constructs were introduced into the genome of BmNPV through homologous recombination and the effect of each deletion on the activity of the polyhedrin promoter was evaluated by measurements of CAT enzymatic activity in extracts of tissue culture cells infected with the corresponding recombinant BmNPVs as well as by primer extension assays. Removal of the entire leader region and eleven adjacent residues of the 5'-flanking sequences of the polyhedrin gene results in a dramatic decrease in promoter activity, which, however, remains detectable through CAT activity measurements. Elimination of an additional 30 nucleotides (nt) of the upstream sequences results in the complete inactivation of the polyhedrin promoter. The functional characterization of a deletion mutant lacking 41 nt of the 5'-flanking sequences has demonstrated that no functions necessary for viral infectivity, replication or assembly are disrupted by this deletion, since the corresponding recombinant viruses propagate in the cells with the same kinetics and to the same extent as wild-type BmNPV. As a result of the deletion mutagenesis, two classes of transfer vectors have become available. The first class can be used for introducing into the viral genome foreign nucleotide sequences under polyhedrin promoter control, while the second one can be used for obtaining recombinant viruses harboring foreign genetic material in an environment which is devoid of polyhedrin promoter activity.     

苜蓿尺蠖核多角体病毒(AcNPV)DNA的提纯及多角体蛋白基因片段的克隆

本文报道了从被AcNPV感染的大蜡螟中提纯到AcNPV DNA,以质粒pBR325作为载体,从AcNPV基因组DNA EeoRI片段克隆中得到含AcNPV多角体蛋白基因片段的重组质粒。经原位杂交,酶切鉴定,DNA顺序分析,并与Hooft Van Iddekinge等人测定的结果比较,证明筛选到的7.3kb EcoRI片段包含了AcNPV多角体蛋白结构基因及其启动基因。核多角体病毒多角体蛋白启动基因是迄今为止在真核细胞中所发现的最强启动基因之一,是理想的表达外源基因的启动子。     

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.     

Firefly luciferase, synthesized to very high levels in caterpillars infected with a recombinant baculovirus, can also be used as an efficient reporter enzyme in vivo

Trichoplusia ni and Spodoptera littoralis larvae were infected with a recombinant AcNPV, having the viral polyhedrin gene replaced with the cDNA encoding firefly luciferase. Both S. littoralis and T. ni synthesized very high levels of luciferase representing greater than or equal to 25% and greater than or equal to 15%, respectively of the total Coomassie blue stainable protein. Luciferase was apparently not secreted into the hemolymph but was contained within the body tissue. Expression in S. littoralis larvae suggests that luciferase can be an excellent reporter enzyme to study virus infection, dissemination and expression in different tissues, host range determination, insect physiology and also to monitor the release of recombinant virus in the environment when used as a biocide.     

家蚕BmNPV多角体蛋白与外源蛋白共包埋入多角体的研究

为了探索家蚕核型多角体病毒多角体的包装特性,构建了一种不形成多角体但能大量表达绿色荧光蛋白(EGFP)的重组病毒vBmBac(polh-)-5B-EGFP,将其与野生型BmNPV共同感染BmN细胞,于荧光显微镜下观察到EGFP与多角体可以在同一细胞中同时表达。从感染的BmN细胞中收集纯化多角体,观察到多角体能被激发出绿色荧光,进一步利用Western blot证实多角体中含有EGFP。上述结果表明,多角体可以将自身病毒粒子以外的其他病毒粒子的成分包装进入多角体,表明多角体的包装机制中存在非特异性识别机制。     

A two-stage bioreactor system for the production of recombinant proteins using a genetically engineered baculovirus/insect cell system

A two-stage bioreactor scheme was developed for the large-scale production of recombinant proteins using a genetically engineered baculovirus/insect cell system. The first bioreactor was employed for cell growth and the second for cell infection. Silkworm Bm5 cells were infected with a recombinant baculovirus, BmNPV/P5.cat, containing a bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the polyhedrin gene promoter of Bombyx mori nuclear polyhedrosis virus (BmNPV). This recombinant baculovirus has been used as an expression vector for the production of recombinant CAT enzyme. A specific productivity of 82 to 90 mug CAT/(10(6) cells) was obtained using the BmNPV/Bm5 expression system, a yield similar to that achieved using the AcNPV/Sf expression system. Repeated infection of high-density cell cultures did not reduce the specific productivity of the CAT enzyme. Most importantly, the problems associated with the infection of high-density cell cultures were resolved by means of controlled infection conditions and appropriate replenishment of spent culture medium following infection. The glucose uptake rate by the cells following infection was 50% higher than that by the cells before infection. Not only did the infection of high-density cell cultures result in consistent yields of 250 mg/L of CAT enzyme, but also the two-stage bioreactor system was proven to be reliable for a long-term operation beyond 600 h. (c) 1993 John Wiley & Sons, Inc.     

The use of defective Bombyx mori nucleopolyhedrovirus genomes maintained in Escherichia coli for the rapid generation of occlusion-positive and occlusion-negative expression vectors

A system is described for the rapid generation of Bombyx mori nucleopolyhedrovirus (BmNPV)-based expression vectors. A series of novel BmNPV genomes, that include a mini-F replicon and therefore can be maintained in Escherichia coli, have been generated. These genomes lack a portion of the essential ORF1629 gene and cannot replicate independently in insect cells. However, they can be used as parental genomes for the generation of expression vectors by cotransfection with a transfer plasmid that includes an intact ORF1629. Only recombinant viruses that have acquired the ORF1629 gene from the transfer vector, and have therefore also acquired the foreign gene of interest, can replicate after cotransfection. Parental genomes with and without a polyhedrin gene are described, enabling the generation of occlusion-positive and occlusion-negative recombinant viruses. Occlusion-positive expression vectors enable the oral infection of B. mori larvae and can therefore be used for the mass production of a foreign protein in infected insects.     

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.