JPH07500962A - Viral vector-based insecticides and expression systems - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Viral Vector-Based Pesticides and Expression Systems The present invention relates to expression systems and biological pesticides. The present invention relates to agents, particularly those containing recombinant viruses.

In recent years, the identification of insect viruses and the use of these viruses as insecticides has Significant research efforts have been devoted to the development of Commercial use of unprocessed baculovirus Large-scale production and distribution have already been achieved, but wild-type viruses cannot kill pests. The relative speed at which the insecticides As a result, the Currently, there are ways to genetically manipulate virus isolates. have been used to increase the virulence of insects, most commonly to improve the metabolic function of insects. Exogenous genes that encode products that are rapidly inhibited or destroyed by the viral genome. It involves merging into a group. The resulting recombinant virus is highly effective and It persists indefinitely at the location where it is released into the environment and spreads from there.

Although the safety of the drug has been fully confirmed, it has been genetically modified. Humans are not responsible for the release of any living organism into the environment. are of wide and considerable interest (Tarrant, 1991). . As a result, future conditions for their use may include "cancellation" or " It is thought that having a built-in "self-destruction" mechanism is included as a necessary condition. This mechanism is , the inability of such genetically modified microorganisms to persist indefinitely in the environment. is designed to ensure that Furthermore, if the virus is non-persistent, the target poses minimal risk to geographically distant, susceptible species that are Probably not.

It is also recognized that there is a need to develop non-persistent viruses; It would be desirable to provide such non-persistent viruses suitable for use as insecticides. This is the object of the present invention.

The Baculoviridae family includes members of the nuclear polyhedrovirus (NPV) group. However, they are found only in arthropods and commonly infect caterpillars. and kill life. These are not known for humans, other vertebrates or plants. are unrelated to any viruses present, and therefore they are specific biological insecticides. is an ideal candidate for the development of

Within the order Lepidoptera, the host range of individual NPV isolates is different and cannot be found in a single host. Some are strictly localized, while others can infect limited areas of multiple species. Ru. However, in all cases, infection of the host is caused by food contaminated with the virus. Caused by ingestion. After entering the intestinal cavity of the caterpillar, infectious virus particles The proteinaceous occlusive bodies (polyhedra) that surround and protect the Rus particles can initiate infection of cells in the midgut arm. Hedron cavity through cell membrane From this initial infection site, the virus particles enter the host via alternative types that begin to grow inside the host. The virus further spreads to other lesions. This alternative particle type is occluded and becomes a polyhedron. but immediately begins to infect other cells within the host; this cell-cell then The infection cycle is repeated many times within each host. In the final stage of infection, polyhedral Particles that are occluded can be produced again and spread to another host.

Similar to NPV, entomopoxvirus (EPV) is also an infectious It exists in proteinoid bodies (spheroids) in the form of EPV is also a recombinant It has favorable characteristics for development as a virus insecticide. These include beetles and bats. baculoviruses (which are not generally infected by baculoviruses) It is a DNA virus that infects pests.

The stability and persistence of NPV and EPV particles and the concomitant nature of virus spread in the field. Capacity is the ability to completely follow the occlusion of this particle into a polyhedron or spheroid. Virus particles that do not survive are destroyed by environmental factors such as desiccation and exposure to sunlight. are highly susceptible and do not play an important role in horizontal virus transmission. Possible (Grandos) and Williams (11+11 iams), 1986, for a review).

Therefore, in a first aspect, the present invention provides that the virus Provided is an insect virus characterized by having a reduced ability to occlude particles. do.

In a second aspect, the invention provides a method for limiting the persistence of insect viruses, which The method occludes the virus particles that the resulting modified virus has. The genome of the virus should be modified to ensure that its ability to Including changing the nom.

The insect virus that is altered may be a wild type, mutant, or recombinant virus.

by any of the techniques common in the art, including recombinant DNA techniques. The genome may also be altered.

In a third aspect, the invention provides for the whole or all of at least one insect virus genome. an occluded virus particle comprising a portion, the occluded virus particle comprising: following its ingestion by the insect host to produce another occluded virus particle To provide occluded virus particles characterized by causing viral infection without .

Thus, the invention according to its third aspect provides the ability to cause only a single field infection of insects. Provides powerful virus particles. Inside the host insect, this virus particle Because it does not have the ability to cause production, it is necessary for horizontal transmission to other insects in the field. This stage (occlusion of infectious particles) is impossible. In this way, the virus according to the present invention A single use of a particle causes a single killing and then disappears from the location. do.

The occluded virus particles of the invention include all or part of a single viral genome. It is preferred that the present invention be It is explained as follows. However, occluded viruses containing multiple insect virus genome types Viral particles may have useful advantages over single genome-type particles should be recognized. For example, the insect target range of occluded viruses is It may be possible to spread the virus by using insect viruses with different specificities.

Therefore, such multi-genome particles are considered to be within the scope of the present invention. , the preferred embodiments described below apply equally to such particles. should be understood.

Insect virus genomes suitable for the present invention may be DNA or RNA.

Preferably, the genome is a baculovirus and an entomopoxvirus. , occluded reovirus.

More preferably, this genome is NPV (nuclear polyhedrovirus) baculovirus. Baculovirus, GV (granulosis virus), or genus A, BM and 0. It is from the entomopoxvirus. This insect virus genome is recombinant It may be a native viral genome or may be otherwise mutated.

As a result of recombination and/or mutation, this insect virus genome becomes closed in the host cell. It should be impossible to cause the production of blocked virus particles. Like Alternatively, this may be achieved by disabling the production of the insect virus's blocking protein. will be accomplished. In the case of nuclear polyhedroviruses, this means the generation of PH-viruses, i.e. N PV is a functional copy of the polyhedrin (PH) gene This entails ensuring that it does not have any Regarding entomopoxvirus, this This is the generation of 1J'1 virus, that is, EPV is spheroidin (LLb,) gene. This involves ensuring that no one has a functional copy of the gene.

To increase the efficacy of occluded virus particles, recombinant insect viruses one or more nucleotide sequences encoding substances harmful to the genome, Examples of such substances that may be contained within the biological domain include, for example, Insecticidal compounds originating from or from the venom of wasps and scorpions, insect hormones , or Bacillus thuringiensis sis) δ-toxin. If the insect virus genome is PH- or LLh- In cases where such exogenous sequences under the influence of the gin promoter, the PH site or the IIL upper site of the virus (respectively). ) is preferably arranged.

In a fourth aspect, the invention provides for the preparation of occluded viral particles according to the third aspect. provides a method for: inducing occluded cells in a host cell; Encrypts at least one viral protein essential for producing virus particles preparing an insect virus lacking a functional gene or gene group, said at least one viral protein that is not encrypted by said insect virus; providing a transgenic organism capable of expressing the protein, and Infect or transfect transgenic organisms with the above insect viruses. transfecting.

The transgenic organism may be an insect, but more preferably the transgenic organism Transgenic organisms are cultured cell lines derived from the tissues of susceptible insect hosts. [For example, clones of Heliothis such as 2D2] tea strain (Dall et al., in preparation)].

Preferably, the transgenic organism has a transgenic protein that encodes an occluded protein. transgene (e.g. PH+ or L41), this insect The virus is deficient in its similar gene (eg, PH- or LpLh-).

From the evaluation of promoter activity, hydrated protein radius and occluded body volume, economic level In order to synthesize enough proteins to produce virus particles occluded with , genes (group C) essential for occluded virus particles, polyhetrin gene) Up to 100 functional and expressible copies required in transgenic insect cells It is possible that “Illegal Recombination” (Low), 1988 ), a large number of plasmids undergo plasmid transfection. After transfection, it is stably integrated into the cell genome. Many copies were consolidated Cells are isolated by applying extreme selection pressure. transgenic thin Strategies to increase polyhetrin gene expression in cells include selectable and amplification Introducing this gene in such a way that it binds to possible marker genes. That's possible! included. Examples of such selectable and amplifiable genes include the enzyme dihydrogen Encoding 07t rate (dihydrofolate) reductase (DHFR) (Kaufman, 1990). Methotre As a result of stepwise selection to increase resistance to xate, the DHFR gene is amplified along with that encoding polyhetrin. enough time has passed Afterwards, the amplified gene is stably integrated into the cell's chromosomes, allowing further selection. There's no need to continue.

Following amplification by this method, up to 200 0 copies were shown to be present in cells (Vur+s et al., 198 6).

Development of suitable transgenic host cells required by the present invention can be accomplished using vectors. This vector is preferably carried out through transformation of cells containing: is essentially active in the host cell and is of host genome or foreign origin Promoter elements derived from and controlling the expression of i-mediate-earlF 1 (r E-1) Pro motor) - suitable for use as a selectable marker for cell transformation Any gene that produces a product (e.g. neomycin from transgene tn5) synphosphotransferase [neomycin resistance] gene). These two The combination of elements is combined with - a sufficient level of expression of: activity is essential or or are regulated by viral infection of host cells, or both. A second promoter arrangement (e.g. Ha plO promoter element or polyhetrin from NPV) - Genes encoding products required for the formation of viral occlusion bodies (e.g. HaNP Polyhetrin) or gene group encoding genes from V.

Particle bombardment, virus- or bacteria-mediated transformation, direct injection transfection, electroporation, and liposome-mediated transfection. Vectors such as the above transform the host cell genome by any transformation mechanism involving It may also be introduced during the system.

In yet another aspect, the invention provides proteins and/or polyhedrons of HaNPV. An isolated DNA molecule containing the code for the phosphopromoter is provided.

As mentioned above, the polyhetrin gene is a non-essential region of the baculovirus genome. It is. Therefore, the desired exogenous genes (e.g. for the insect harmful substances listed above) HaNPV or other viruses by homologous recombination. with the HaNPV polyhetrin code to integrate with the Curovirus genome. DNA molecules (e.g., transfer vectors) containing sequences and/or promoters may also be used. good. This exogenous gene may be promoted by its native promoter or by any other suitable promoter. more preferably a strong HaNPV polyhedrin promoter. It functions and connects to the data. In this way, the present invention utilizes polyhetrin encryption. and/or comprises an exogenous nucleotide sequence(s) located within the promoter region. It should be understood that this also extends to recombinant HaNPV PH-viruses.

In yet another aspect, the present invention provides proteins and/or proteins of HaNPV. An isolated DNA molecule containing the code for the -1 promoter is provided.

In yet another aspect, the present invention provides a method for controlling the proliferation of pests in a place where the pests are infested. The present invention provides a method for controlling occlusions according to the present invention. Including the use of insect virus particles or insect viruses in this location. The virus or occluded virus particles form an admixture with an agriculturally acceptable medium. are doing.

The present invention will now be further explained by the following figures and non-limiting examples.

n Otori 1 Gen 1 Tomo Figure I shows the nucleotide sequence of the HaNPV polyhetrin gene. This Nucleo The sequence of a putative polyhetrin protein originating from the tide sequence is shown in Figure 2. Part of HaNPV IE-1 gene obtained by PCH described in Example 1 The putative IE-1 tag originating from this DNA sequence shows the nucleotide sequence of Protein sequences are shown.

Figure 3 Nucleus between baculovirus IE-1 genes at the start site for transcription The 0pNPV sequence shown, showing homology to the leotide sequence, is Teilmann (Th Also from Eilmannl and Stewart (1991) The sequence of AcNPV was published by Guarinol and Su- mers) (1987) - [GS] and Chisholm and Henner (1988)-[CH] to maximize homology. A gap was introduced to Nucleotides shown in uppercase letters are all This is common to viral sequences. Nucleotides that differ between these sequences are shown in lowercase letters.

The underlined nucleotides correspond to the transcription start site identified as the IE-1 gene. I am responding.

In the HaNPV array, the numbers shown in bold within the same block (1 block) cleotide is present closest to the transcription start site identified as AcNPV. It is something that

−■ Baculovirus and especially HaNPV (heliovirus, a major pest of a wide range of crops) Heliothis is a suitable software for demonstrating the present invention. It is a virus. Because Shieh (1989) and Vla k) Insects cultured according to methods reviewed by (according to publications) and Insect larvae grown in large quantities or in insect cells can be easily 6 Both methods have distinctive advantages and For example, large amounts of untreated NPV can grow in larvae, but their survival Difficulties associated with material handling, maintaining a contamination-free growing environment, and downstream product processing. However, this production process is highly disadvantageous and is highly pathogenic. It is expected that the level of recombinant NPV will simply fall too low before killing the host. and reduce the product yield from this process.

Conversely, the virus yields already reported for cell culture systems are relatively low. However, this is because 'raw' viruses are more expensive.

Nevertheless, evaluation of the effectiveness of alternative systematic expression and control of product quality is essential. control is clearly facilitated by this generative strategy. Therefore, in Example 1 The methods and products presented were based on the use of exclusively cultured insect cells. field Live HaNPV was produced by Dr. R. Teaklel (Queenslan). Department, Of, Primary, Industries (Queensl) and Department of Primary ), 0 artificial food obtained from Indooroopilly (Indooroopilly) Haliothis armigella, 5 days old after being fed with food. ra) larvae (Tiekle and Jensen, 1985) and differential This wild-type isolate was cultured twice with polyhedra purified by centrifugation. Ta.

-V Mar- A gene with a polyhetrin code (PH), a promoter that binds to it, and both wild-type H. amigera NPV (H A 7.3 kb Xhol-F fragment of genomic DNA from p To obtain A44NSl, pTZ18U (Bio-Rad) of the plasmid derived (through previous BamHI digestion and end-filling) from 5alI site. The site that commands mutagenesis is originally 5'AT Positions including and around the PH translation initiation codon, read as GTATAC3'' The viral sequence was used to replace 5'AAGGATCC3°.

This mutagenesis removes the translation start site of PH and creates a BamH1 region in the fragment. Digestion of sBamHI that introduced the HaNPV position and end-filling of this construct Under the control of the promoter element of PH, Drosophila esterase-6 (LLJL-6) was generated. Digestion of EcoRI, and A cloned DNA segment originally of Drosophila origin t) end-filling for cloning into the mutagenized pA44Ns1 vector. The LLJL-6 sequence was given as a match for this. This is pA44Ns1/Shoji Normal methods of molecular biology to obtain LIti-6 (e.g. (See Sabrook et al. (1989)).

Original genomic HaNPV DNAj3 and purified pA44Ns1/Sho Cotransfection of D. melanogaster 11-6 n) resulted in viral infection of susceptible cultured host cells. stop and the sequence of replicating viral DNA and I) A44 NS 1/Drosophila fJ. Subsequent homologous recombination with viral sequences on jL-6 resulted in recombinant A44E. 6 HaNPV (HaNPV PH-est”) was generated. The presence of a functional copy of the gene is detected by enzymatic assays, and the recombinant isolated using standard microbiological methods.

Go to 2.1ゝ1ゝ　-Rohniゝ The polyhetrin gene is derived from wild-type HaNPV viral DNA as a template. It was specifically amplified by PCR (polymerase chain reaction). ply used Primer is: SRI: TATGAAGATATCTGTCGT (at the 51st end of the promoter) ) Polyexit TATTTATTGAAGATAACTT G (at the 3° end of the gene) The oligonucleotide named SRI is located at the start codon of the polyhetrin gene. Located 140 nucleotides upstream of the 5'-untranslated leader of polyhetrin mRNA -, transcription initiation site, and promoter.

Polyexit is polyazonylation. signal, located 270 ntS downstream of the end of the polyhetrin gene. PCR is , the expected size is 1.15 Kbp), which is the vector -pIElneO (Jarvis et al., 1990) This is the plant end cloned into. It cuts at the unique EcoRI site. cleaved, end-filled with Frenow fragment, and dephosphorylated. inserts Three recombinant plasmids carrying (1nserts) were selected: These were named clones 2.3 and 9. HaNPV polyhetrin gene The array of I! Shown in II.

■-Kuchi 12ゝ The IE-1 genes from two baculoviruses have been sequenced. These are 0 pN PV (Theilman and Steward, 1987) and also from AcNPV. According to Garino and Summers, 1987 (hereinafter referred to as O5), According to two groups, Chisholm and Henner, 1988 (hereinafter referred to as CM).

Comparison of sequences from two viruses reveals the nucleotide level differences between them. It was shown that there is very little homology. Three important things about conserved sequences blocks were revealed: two sequences were synthesized for these regions. In addition to the primer sequences shown below: a) In the promoter, approximately nt, -5001, 0pNPV 5' from the start of transcription To TGCGG-CCAC8TC'rrc 3'AcNPV (GS) TGC to GC-CGAC8τT frGTAcNpV (CH) TGCGCGCGACA TTTrTGT primer H, a IE IA: 5'-TGCGG, -, CCACATCfff'GTb) in the middle of the gene op! GAGTACACTAACM? TACTACATW, GACAATCGCGTG TITGTGG? Primer HAIEIB (complementary to above) ATGTACCAT CTGTrAGCGCACMACACCA-5' primers HalEIA and H PCR with aIEIB detected the corresponding segment of the E-1 gene from HaNPV. was used to specifically amplify the target. The sequence of the 965bp PCR product is shown below. Shown in 1. This gene segment corresponds to the IE-1 gene described above. It is approximately 400 bp shorter than the region in which these two sequences share limited sequence homology. It was shown that it is short-lived. There is significant sequence homology around the transcription start site, and their This is also where the homology between other IE-1 genes extends the longest (Figure 3).

The first methionine encoded by the HaNPV amplicon is located 92 nucleotides downstream of G, marked in bold. in that array This G shows homology to other NPVs around the transcription start site, but AcNP V corresponds most closely to the nucleotide that initiates transcription of the IE-1 gene.

The open reading frame starting with this first AUG interferes with the rest of the PCR-amplified sequence. .

The IE-1 gene of other NPVs is very easily translocated during the viral replication cycle. These promoters can constitute activity within the cell (Jarvis et al., 1999). 990, supra), their transcriptional activity is actually more complex, and their replication For 0pNPV, IE-1 The gene product is autotransactivated in some cell lines. It has been shown that auto-trans-activation is possible. Illman and Steward, 1991).

Therefore, we believe that the genes we identified by PCR amplification are indeed in the early stages. An experiment was conducted to demonstrate that the transcription is possible. Infection 11k (p, i,), different Northern plot of RNA from HaNPV-infected cells obtained at different stages (b lot) were investigated using cloned PCR products. This is seiza 8 hours pi.

showed evidence of early transcription, with maximum values at 48 h p.i. (the last stage tested). increased towards. Similar observations were made using the 0pNPV IE-1 gene. (Theilmann and Steward, 1991).

゛ Ma Bi゛ A probe made from the cloned PCR product was used to detect the putative I For detection of restriction fragments of the entire HaNPV genomic DNA including the E-1 gene It was used. This blow 1 was hybridized to the following restriction fragment: Eco RI fragments A and L or M, the 8 kb EcoRV fragment, and H indII fragment D, EcoRI and HindIII fragments are all It has been completely mapped (■apped). Therefore, this gene is HaN The complete map of the PV genome (occupying approximately 81 map units on the ++apl) and obviously two adjacent EcoRI A and L or M fragments. forming a link between components. Through precise mapping, this gene It was shown to be located on a 2kb DraI fragment within the region.

In this example, the rE-1 promoter was used, but other active constitutive promoters Romotors are also suitable. For example, 11 Yu Jl (Rourkel and actin promoter of yeast (East, unpublished) or IL-su 8pp −Promoters of other genes that are abundantly expressed in be. The strategy for obtaining these promoters is as follows. unselected abundance in cells Create cDNA clones for specific mRNAs and then clone from unselected abundant cellular mRNAs. When crossing A into the Northern plot, select the cDNA that gives the strongest band. Choose. Next, this cDNA is used to create a genomic library. and identify associated promoters. More transient expression if candidate promoter If the appropriate fragment with the desired activity is selected by analysis, Used to drive selectable genes in expression plasmids.

Using the three plasmids described above (from clones 2.3 and 9), DOTMA test chestnut (Boehringer Mannheim) 1JIBCIRL-H2-AMl cells (Pine Kintrash (McLnt.

sh) and Ignoffo (1981). -evening After transfection, medium and 10% FC3 were added and left for 24 hours. . Next, add 0418 (Sigllml) to the medium containing 10% FC3. Add so that the final concentration is 1 mg/ml or 0.5 mj/ml and perform screening. Ta. 4.5 ml of the filtered and sterilized mixture was added to each transformation mixture. each of the two Three cultures were grown at antibiotic concentrations. After 2 weeks, replace the media once. , the selection pressure was removed to allow surviving cells to grow. 0.5mg/ml 04 Only cells exposed to 18 survived and grew. 6 weeks of growth without culling pressure After this, samples of cells containing each of the three transforming plasmids were collected and - It was stored frozen at 196°C.

Neo resistance selection methods have been shown to have poor cell recovery and growth, making them difficult. It was proven that. Yet another difficulty was encountered using plasmids #2.3 and 9. This also occurred when evaluating the production of polyhetrin from cell lines created using

Therefore, future PH" host cell lines could be created using plasmids containing: There will be.

- Driven by AcNPV IE-1 or HaNPV IE-1 promoter selectable genes (e.g., short engineered genes or amplifiable HFR genes) ;as well as - Polyhedrin promoter and gene.

Such plasmids can be prepared by one method, or by electroporation or plasmid production. Can be used to transform cell lines through alternative transformation methods such as transformation. I can be there.

When a PH cell line is infected with a virus whose genome lacks the polyhetrin gene, Production of polyhetrin from the transgene in the cell line should be induced .

Infection of transformed H2 cells containing PH inhibits the growth of recombinant HaNPV PH- Viral growth in cultured host cells using virus particles initiated by involves exposure of transformed cells to a liquid inoculum containing infectious particles derived from be caught. An alternative means of infection is the use of viruses purified from virus envelopes. Exposure of cells to particles and injection or transfection protocols ( involves introducing infectious genetic material into cells through .

Conclusion 1 Until now, virus replication without the formation of polyhedra and accumulation of polyhedra without viruses have been reported. (the latter also in the absence of virus) was observed. Both results also Not reproduced.

These results suggest that polyhetrin expression may be due to one or more of the following reasons. This is believed to reflect preliminary difficulties in the temporary coordination of

1. HaNFVPH-infection leads to abort of host protein synthesis, whereupon In some cases, the polyhetrin protein is not expressed at all.

This phenomenon occurs in baculovirus infection, especially in the later stages of virus replication (12 −18 hours pi, i.e., following replication and extracellular (non-occluded) virus (occurring simultaneously with the formation of gas). Cessation of host protein synthesis occurs in late stages. From the action of virus proteins and the unique active RNA decay process of host RNA This seems to result (Ooi and Miller). ), 1988).

2. Location where the polyhetrin transgene is present on the chromosome of the host cell The effect is due to the RNA polymerase responsible for transcription of the late NPV gene. Not transcribed by position 3. Significant basis of expression from the polyhedrin promoter in virus-free cells (Constitutive) level.

Friesen and Miller (1985, 1986) We detected early polyhetrin-related RNAs such as ,i. But low level configuration This expression occurs only 2.3 hours before cells begin to make large amounts of polyhetrin. This is not expected to be a problem in normally infected cells, as this would occur. child In contrast, PH''' transgenic cells can be used for long periods of time (i.e. until infection) allows polyhedrin to be expressed at low levels from the polyhedrin bromomotor. It is. Accumulation of polyhetrin and concatenative formation of virus-free polyhedra occur in cells. Viability and/or virus replication may be affected.

Therefore, the generation of occluded virus particles can be achieved by adding one or more of the following methods to the above method. This can be easily achieved by making the above modifications.

(A) Late viral protein that is responsible for stopping Iil main protein synthesis. Inactivation of encoding genes.

This variant identifies the viral gene(s) that cause the effect and then blows this gene. Mutagenesis of (group) to stop its activity or make its function temperature sensitive do. This gene can be detected by any of a number of standard techniques to detect irregularities in the viral genome. Virus-induced arrest can be easily identified by several methods, including mutagenesis. Validation of the block (preferably this block is temperature sensitive) follows. valid Selectable marker transgenes transcribed from constitutive promoters ( For example, at any given point, constitute a transgenic cell line that carries (neo) This can be achieved by applying neo-selection (e.g. during baculovirus infection). can be done. Any virus that can induce host protein shutdown , therefore preventing the cells from developing neo-resistance and thus causing them to die. wax; a virus-replicating cell that is unable to induce host protein arrest With this strategy, only the cells would be able to express resistance genes and survive. is probably highly diluted to ensure infection by only a single virus. This may require a standard infection known as end-point dilution. This will be accomplished by steps.

In this variant, candidate genes for inactivation are detected at a later stage (approximately 10 h p , i., Friesen and Miller, 1986 Wilson and Miller. Virus-induced 3-proteins observed to bind to chromatin It encodes a 9kd protein. Such binding may lead to host gene transcription. may be responsible for the cessation of host protein synthesis through a direct negative effect on child This suggests a direct and easily testable research method: determining the appropriate viral genes. transgenic cells that mutate to be temperature sensitive and produce occluded viruses. selectively inactivate functions in genic cells.

Temperature-sensitive mutations that are unable to transition to later stages of the viral life cycle There is a description of the body (Miller et al., 1983. Gordon) and Carstens, 1984).

Recombinant NPV according to this modification can serve as an expression system for useful exogenous genes. It can also be useful.

(B) Providing host cells with the transgene in an extrachromosomal location.

The major very late proteins of AcNPV, both plo and polyhetrin. The promoter for It has been shown that when present on Smid, it exhibits virus-inducible activity (Nebe Knebel et al., 1985, Rankin et al., 198 8, and Qin et al., 1989). These discoveries were made at a very late stage. The virus gene does not actually need to be present on the viral genome to be active, and Located on a plasmid or engineered minichromosome that can replicate in insect cells transgenes that are capable of escaping virus-induced shutoff of host protein synthesis. It has been suggested that polyhetrin can be produced by Such expression systems are Mother (derived from 2 micron ring; Romance (Ro++anos) et al., 1992) or or plants (viruses, such as geminivirus) Already for Kanevski and +992) being developed.

This research method could also solve the problems listed in (2) above. . The reporter gene is inserted into the virus into a transcribable plasmid. inserted under the control of the polyhetrin promoter and then transformed into the host by illegitimate recombination. Insertion into the chromosome and expression before and after infection with wild type or PH-HaNPV By comparing the RNA polymers responsible for transcription of late viral genes, A simple test to test whether the enzyme can transcribe genes on the host chromosome It can be carried out. If expression from the transgene in the chromosome of the cell is observed Without it, virus replication would not have access to virus-induced RNA polymerase. It will be necessary to explore whether this is the cause of transgene generation.

A simple experiment can be carried out to generate a successful promoter from a transfectable plasmid. will include expression of the gene for virus-induced RNA polymerase.

Again, first in the transfectable plasmid and into the host chromosome by blowing. The reporter gene (e.g. The expression of GUS) is a protein that expresses the gene for viral RNA polymerase. A comparison will be made with the lasmid before and after transfection.

Instead of placing the transgene on a plasmid or minichromosome, virus, using translocation or inducers based either in the bath or elsewhere. A chromosomally located transfectant excised from a chromosome at the appropriate stage of replication. It may have a horn. If there are enough copies of the chromosome, the polyhetrin gene can be It becomes possible to use a sufficient number of '11M plasmid-like DNA groups to be retained. Poly Any ebisomal DNA that carries the hetrin gene is susceptible to recombination and its attendant To avoid the risk of reinserting the polyhetrin gene into the virus, It would be necessary to retain little or no sequence homology with Rus. . Weyer and Posseel (1987), p. transfected plus that retains the lo-promoter/CAT gene configuration between the mido and the virus (which actually also carries the p10 promoter) in the infected cells. It is noteworthy that we did not find any evidence of recombination.

(C) PH using a mixture of full-length genome virus and defective virus” Transfection of host cells.

Lee and Krell (1992) For further cultures (up to 81 in total), viruses contained in extracellular virus particles Most of the DNA is derived from AcNPV map units 85.0 to 87.2. A deletion gene consisting only of a few highly repeated 2,8 kbp fragments It was observed that it was in the form of a gnome. This segment is used during virus preparation. All necessary for replication is apparently provided by the small amount of full-length genomes that exist. - together with the E. virulence-acting factor, sufficient for the replication and packing of the viral DNA. , contains important cis-acting elements. At this stage of the culture, all of the produced The virus is in extracellular form; infected cells are mostly infected by day 3 p, i. No cells produce polyhetrin, only some cells show some cytopathological effects showed that.

These observations are mainly due to the presence of a small amount of defective genome viruses and a small amount of full-length genome viruses. mixtures that do not induce any cessation of host gene expression or intracellular viral We show that it is induced only at late stages in the replication cycle. polyhedron The lack of production is likely due to loss of the polyhetrin gene from the defective genome. This is caused by And this defective genome has most of the available for transcription. It consists of viral DNA.

Such a defective genome would be a suitable vector for toxin genes, and , which should make it possible to achieve the generation of polyhedra containing primarily defective viruses. not present. If a full-length genome is used, it is possible to There should be.

(D) Driving of the PH) transgene by a virus-induced cellular promoter Movement.

A cellular gene, 5F21, can be activated by baculovirus infection. Discovered (Mainprize, etc.), unpublished, flexible (as cited in Fresan and Miller, 1986). in this way, In this variation, the host cell is infected with 5F21 or other virus-induced cell proteins. Substantially the above except for the polyhedrin promoter which is replaced by the promoter may be transfected with vectors such as The gene clones the DNA for mRNA into the desired very late stage of viral replication. The virus is induced at step 1 and then selected by probing for viral DNA. It can be isolated by Any cD that does not hybridize to viral DNA NA probe is selected.

To prevent any potential virus-related RNA degradation problems, such viruses An inducible cellular promoter is identified at the 5° end of late baculovirus mRNA. Preferably, it is modified to include a continuous arrangement (Miller, 1988).

(E) Suppression of polyhetrin transgene transcription by promoter occlusion.

The basal (constitutive) level of transcription is the difference between transitional expression studies and transgenic cell experiments. can be easily explored using reporter genes (e.g. GUS) in both can. If the basal level of transcription is significant, then this transformation requires polyhetrin transcription is repressed through promoter occlusion or some other mechanism. This includes placing the host gene within suitable proximity to the host gene. This suppression is Its function ceases when host gene transcription decreases at late and very late stages of infection. Stop.

(F) Transcription of PH' host cells using PH-HaNPV at low MOI Sfect.

The rate of decrease in host protein synthesis depends on the multiplicity of infection (MOI). M.O. The higher the I, the faster the host protein decreases compared to the lower Mol (Marni Maruniak and Summers, 1981), therefore our strategy to obtain the lowest possible titer of occluded virus. It is preferable to determine the MOI that is possible. Whether natural or due to deformation, low After infection at MOI, it replicates rapidly and accumulates large amounts of polyhedra at later stages. A virus that can do this would be able to overcome this difficulty.

Other possible variants include splicing at very late stages of NPV infection. This includes taking advantage of apparent reductions in capacity. Polyhetrin mRNA release The donor sequence can be deformed to combine splicing donor sites and The sequence of the rehetrin open reading frame is transformed in a non-deletion manner to form a splice acceptor. part can be formed. This strategy also uses the generate other sequences (e.g., the lasso region of the splice acceptor - upstream). It may also be necessary to make other minor transformations to (Padgett et al., 1986). These changes to the polyhetrin gene As a result, splicing occurs in uninfected cells and early in infection. Destroys polyhetrin mRNA from to late stages, but very late stages It won't be destroyed.

2 凰り9ヱ The occluded virus particles of recombinant HaNPV are present in infected cells. The induced lysis releases insoluble cells or lysed cells into the culture tissue medium. May maintain connections with cyst fragments. The liquid medium is removed from the cultured tissue; Residual cellular material is mechanically or enzymatically separated from the solid support. occlusion will The particles are purified from these raw materials by processes including differential centrifugation and the like.

■ − Like mammalian poxviruses, EPV replicates in the cytoplasm of infected cells. Manufactured. Nevertheless, in transgenic cell lines, trans( For example, the gene (encoding spheroidin) is part of the viral life cycle. It is desirable for the present invention that the protein be expressed in the nucleus at an appropriate stage within the nucleus. This strategy is Recombinant l and 5-EPV to convert spheroidin gene transcription in the nucleus. Molecular switches (e.g., under the control of the late HaEPV promoter, HaNP This includes the expression of the V transcription factor IE-1 gene).

−■　Mar　−−ゝ　Bae -v- Published sequence data (Vialard et al. 1990) were used is used to inject oligonucleotide primers into the polymerase chain reaction (PCR). 6 Next, as a template we used HaEP Using restriction digested genomic DNA prepared from EPv of V), HaEPV spheroid A fragment of the leudin (sph) gene was amplified.

This fragment serves as a template for the synthesis of radiolabeled probes. Southern plotting In the ng) protocol, this alternatively specifies that the sph gene is Size restriction fragments were used to localize. From this work, this The gene is 4.9 kilobase (k b ) B g l It is located in the II fragment and on the approximately 9 kb EcoRI fragment. has been confirmed. This BglII fragment was then cloned and sequenced It was done.

The bacterial reporter gene GUS is a clone between the sph promoter and the gene. By site-directed mutagenesis of the BglII genomic fragment Placed in the created Bam H1 site. The resulting DNA fragments are then The isolated but unaltered genomic HaEPV DNA Alternatively, the DOTMA reagent (Berry Helicoverpa cultured using 1B CI RL-Hz-AMI cells were cotransfected (cotransf ect) or infection/transfection.

HaEPV replication in co-transfected cells is a homologous recombination event. As a result, the part of the structure containing the reporter is transferred to the viral genome. integrate into. In this way, this process replaces the jLJL gene location of the original virus. A recombinant photogenic virus (recHaEPV) carrying the GUS gene at the site is generated.

Expression of the reporter gene is driven by the single promoter and can be expressed using standard enzymatic methods. It will be detected by testing. GUS activity significantly above control background levels If detected, it indicates the occurrence of recHaEPV.

In the final structure, HaEPV has one IE-1 gene in place of the GUS gene. do. This IE-1 gene was obtained using a method similar to that for the GUS gene described above. It is placed from 3' to the spheroidin promoter. This IE-1 gene may further include nuclear localization signals (e.g. from the SV40 T antigen) .

Insertion is facilitated by using PCR primers that retain appropriate restriction sites. It may be done. The resulting plasmids are then cultured as described above. Helicoverpa cells (genomic HaEPV DNA or infectious HaEPV pi) used to contrafect

◆ AcNPV delayed early (DE) The sequence of the gene has been published (Garino and Summers 1986a). this inheritance The offspring are suitable for expressing the coding sequence for sphaeroidin in the host cell. Give promoters. Alternatively, HaEPV or a heterologous sphaeroidin promoter Alternatively, the promoter from the HaNPVDE gene may be used. This HaN The PV DE gene is prepared by designing appropriate primers from the AcNPV DE sequence and It is isolated by amplifying the DE gene by H. This DE gene is particularly The promoter is a homologous repeat sequence on the baculovirus genome. This IE-1 gene product is significantly transgenic when located in close proximity to the copy. have the ability to be activated (Garino and Summers, 1986bi Theilman and and Steward, 1991).

a selectable marker gene and a sequence linked to a DE or other suitable promoter. The structure holding the spheroidin to be encoded is similar to that described in Example 1. used to transfect host cells.

r-ray Recombinant IE-1''l1-) (infection of the two-cell lineage with aEPV induces the production of spheroidin from the transgene in cell lines. culture host a liquid inoculum containing infectious particles derived from prior virus growth in cells Various methods include the use of virus particles that begin to grow by exposing cells to Infection can also be initiated by

N　P　ll’a! Problems similar to those faced may also apply to EPV. . If so, the production of EPV-containing occluded virus particles according to the invention requires The variations of the generation method to be used will be similar to those proposed in Example 1. However, HaEPV can sustainably grow in cells of L. in vitro. , even after 14 days p.i., there are no obvious processes leading to cell necrosis and decay. I won't wake you up.

Therefore, the occluded virus particles according to the present invention are transgenic jLJLh" li, f2 cells (particularly strain BCIRL-Hz-AMI) with 5ph-HaEF can be easily produced without the difficulties faced by NPV. It is expected that

Occluded virus particles may be purified from these sources by methods involving differential centrifugation. good.

1-1 experience Different transgenic host cell lines are used to detect occluded cells produced by host cells. The number of virus particles, the average number of infectious viruses occluded in each particle, and the occluded viruses. Variations occur in the ease with which viruses are released from virus particles.

Those cell lines exhibiting the most desirable balance of characteristics are HaNPV l:5- or Obstructed virus particles produced after inoculation with a standard amount of HaEPVLpH- Simple quantification of the number of progeny and release from the resulting number of occluded virus particles under standard process conditions. Host cell TCIDI of released virus. Selected based on the decision of

Small-scale laboratory and glasshouse tests using target insects can reduce large-scale virus particle production. It also provides important indications of the suitability of the method selected for construction and subsequent field use. do. In these tests, worms were fed artificial food containing occluded virus particles. provides a rapid and sensitive bioassay of viral particle infectivity and non-persistence. vinegar.

needle Chisholm, G.E., and Henner, D.J., 1988. Virol, J. ol), 62:3193-3200 Friesen, P. D., & Miller, L., (1985) J. Villeur, 54: 39 Friesen, P. D., & Miller, L. (1986) Microbiology and Immunology. Current topics, 131: Granados, R. R., & Williams, K. A. 1986, 'Baculovirus. In Vivo Infection and Replication of Baculoviruses Volume 1 Gallino L, A , and Summers, M. D., (1986a) J. Guirol. 57:56 Garino, L, A., and Summers, M. D. (1986b) J. Gwirol. 60:21 Gari No L, A, and Summers, M, D, (1987) J, Virol, 61:2091 Jarvis, D, L, Fleming + tr, o, w, l co Kovacs.

G. R., Summers, M. D., Garino, L. A. (1990) Bio/Techno. Logi 8:950-955 Kanebsky, 1. F., Takur, S., Co. sowsky), L Sunter o, l Brou gh) C1+ Bisaro D, and Maliga P , (1992) Plant Journal 2:457-463 Cow 7? N, R, J. 1990% elementary school method 185, 537-566゜nebe)'yD,,hl<-t (Luebbert) H, + Doerfler (Doerfler) W, ( 1985) EMBOJ4: 1301-1306 Lee H, Y. and Rell, P. J. (1992) J, Virol, 66:4339-430, 1988 Gene. Recombination of materials. Academic Press, New York Marnia) y J, E, &? V-S M, D. (1981) Will xology 109:25-3 Miller, L., , Trimarch R, E, , Browne D-+Pennock G, D, (1983) Virology 1 26:376-380 Miller, L., (1988) Enrub Microbiol. (Ann, Rev. Microbiol,) 42:177-199 Matsukintosh, A. H., and Ignofo G., M. (1981) J., Inbard. Invertebr Pathology 38:365-403 Ooi, B. G., & Mira. - L, Ni, (1988) Virus Ji 166:515-52 Kin J, Liu (L iu) +, + Weaver, R, F, (19119) J.Ken. Virol, (Gen, Virol, 170: 1273-127 9 Rankin, I.C., foi, B., O., & Miller, L., (1988) Gene 7 o: 30 Manos, M, A, llcorer + C, A, and flare (C1are), J, J, (1992) Yeast 8:423-488 Luke and East, unpublished Sampurtsuk, J. et al. (1989) Molecular Cloning Laboratory Manual, Vol. Edition, Cold Spring Harbor Research Press bor Laboratories Press) a Shih, T., R1 (1989) Industrial production of viral horax, 36, 315-3. 43° Tarrant, D. (1991) Weekend Australian Magazine October 1 3-14, pp. 29-40.

Tickle R, E. and Jensen J, M. (1985). Shin (Singh), P., and Moore R, F. (eda) Handbook of insect husbandry. Second Fist, Elsevier, Amsterdam, No. 313-3 22 pages.

Theilman D, A., and Steward S. (1991) Virology 180:492. Vuillard, J., E., Yuen, L., & Richardson, Ri. Chardson), C, D, (1990) J, Virol, 64:580 4-5811 Vlak, J. M. Genetic engineering of baculoviruses in insect control. . 'Molecular Approaches in Basic and Applied Entomology' Publishing, Oakss hott), J-Q-11tfifhitten), M, J, , ads, Springer Verlag, New - York.

Wire U6 and Posse R, D. (1987) Nucleic Acid Res, 16: 3625- 3653 Wilson, M. E., &; Ra, L. (1986) Wills letter 151 :315- Worm, F. M. et al. (1986) Broc National Academy of Sciences ( Proc, Natl, Acad, 5et) USA83.5414-5418 In　/”l　+l’l　m　Ca　rl’t　u(ko International Investigation Report I-Atrocities (Nami-1-nNaPCTIAI+92100413) International search report PCTIAU92700413This eightnexliscsth eknown”A”pubQc3Lionlevelpatentfamily memberrelat Amashi■狽庶Re, paLer+tdocumentsc itcdintheabove-menLi, onalir++ernauon alsearchreport, TheAustrali≠shi oatentOr nce+s+nnowayliable for these p-age cularx wh+ch ue merely given for re purpos e　o(tnror high ≠ floating shield■ Continuation of front page (72) Inventor: Dull, David James, Australia Lian Capital Territory-2602 Dixon Moncrief Street 3 (72) Inventor Gorton, Karl Heinrich Julius Australia Australian Capital Territory 2611 Wes Tonshev Aliar Street 18 (72) Inventor Handzlik, Terry Nelson Australia Lian Capital Territory 2611 Chapman Ghana Brace 3 (72) Inventor: Srisukantha, Aragakorn, Australia Capital Territory-2615 Florima Calum Circuit 31

Claims (39)

[Claims] 1. Reduced ability to occlude virus particles compared to wild-type virus An insect virus characterized by 2. Reduced ability to occlude viral particles compared to wild-type virus A recombinant insect virus characterized by: 3. Baculovirus, entomovox virus, and occluded reovirus 1 or 2, further characterized in that the virus is selected from the group consisting of: is the virus described in 2. 4. NPV baculovirus, GV baculovirus, and genera A, B, and C It is confirmed that the virus is selected from the group consisting of the entomopoxviruses. The virus according to claim 3, characterized in that: 5. occlude the virion contained in the resulting modified virus The viral genetics ensure that the ability is reduced compared to the wild-type virus. Methods for limiting the survival of insect viruses, including altering their gnomes. 6. by recombinant DNA technology, or by chemical mutagens or mutagenic light. 6. The genome is changed by mutation according to claim 5. the method of. 7. The insect virus is baculovirus, entomopoxvirus, or blockage. The reovirus according to claim 5 or 6, characterized in that the reovirus is selected from the group comprising How to put it on. 8. Insect viruses include NPV baculovirus, GV baculovirus, and A Entomovox viruses of Genus B and Genus C. 8. The method of claim 7, characterized in that: 9. occluded virus containing all or part of at least one insect virus genome; virus particles, and this occluded virus particle is responsible for its ingestion by the insect host. Subsequently, viral infection occurs without producing another occluded virus particle occluded virus particles characterized by: 10. The at least one insect virus genome comprises a baculovirus and an insect virus. A virus selected from the group including tomovox virus and occluded reovirus. 10. The occluded virus according to claim 9, characterized in that the genome is from Rus. particle. 11. The at least one insect virus genome is from a baculovirus, In particular, the request is characterized in that it is a genome from NPV or GV baculovirus. 11. The occluded virus particle according to claim 10. 12. The at least one insect virus genome is an entomopoxvirus. , particularly from entomopoxviruses of genera A, B, and C. The opened virus particle according to claim 10, characterized in that: 13. The at least one insect virus genome produces an occluded protein. Any one of claims 9 to 12, characterized in that it is impossible to occluded virus particles as described in . 14. The at least one baculovirus genome comprises polyhedrin (po1 12. yhedrin) occluded virus particles. 15. The at least one Entomovox virus genome is a spheroid. 13. The closed block according to claim 12, characterized in that the closed virus particles. 16. The at least one insect virus genome encodes a substance harmful to insects. Claims 9 to 9 are characterized in that they contain one or more exogenous nucleic acid sequences that 15. The occluded virus particle according to any one of 15. 17. Claims characterized in that they contain all or part of a single insect virus genome The occluded virus particle according to any one of items 9 to 16. 18. Preparation of occluded virus particles according to any one of claims 9 to 17. in a host cell, the method comprising: at least one viral protein essential for producing virus particles occluded with Using an insect virus that lacks a functional gene or gene group that encodes a protein. to mean; at least one virus tan that is not encrypted by the insect virus; providing a transgenic organism capable of expressing the bacterial pathogen; and Infect or transfect the transgenic organism with the above insect virus. transfecting. 19. The above transgenic organism is a cultured insect cell line. 20. The method of claim 18, wherein: 20. not encoded by the insect virus in the transgenic organism. a transgene (tr) encoding at least one viral protein; ansgene) is located at an extrachromosomal location. 20. The method according to claim 18 or 19. 21. The insect virus is a PH-baculovirus, and the above-mentioned transdivirus is a PH-baculovirus. 21. Claim 18, 19 or 20, characterized in that the biological organism is PH+. How to put it on. 22. the insect virus is sph-, and the transgenic organism is 21. The method according to claim 18, 19 or 20, characterized in that it is sph+. 23. The transgenic organism has multiple expressible copies of PH. 22. The method according to claim 21, characterized in that: 24. The transgenic organism has multiple expressible copies of sph. 23. A method according to claim 22, characterized in that. 25. The insect virus mentioned above is a virus that causes host protein synthesis to stop. Also lacking is a functional gene or gene(s) that encodes a protein(s) 25. Method according to any one of claims 18 to 24, characterized in that. 26. Infection or transduction of the above transgenic organisms using the above insect viruses sfection is characterized by a mixture of the entire genome and incomplete forms. 26. A method according to any one of claims 18 to 25. 27. not encoded by the insect virus in the transgenic organism. transgene(s) encoding at least one viral protein as described above; However, cell promoters induced by viruses, especially SF21, It is characterized by functionally acting and binding to the cell promoter, especially SF21. 27. A method according to any one of claims 18 to 26. 28. not encoded by the insect virus in the transgenic organism. transgene(s) encoding at least one viral protein as described above; functionally binds to the late viral promoter and converts the parenthesized transgene into a site in the host genome such that transcription from the promoter is suppressed by promoter occlusion. The method according to any one of claims 18 to 26, characterized in that the method is located at Law. 29. Infection or transduction of the above transgenic organisms using the above insect viruses A claim characterized by a low multiplicity of infection (MOI) of infection. The method according to any one of Items 18 to 28. 30. HaNPV Polyhedrin Encrypted Region and/or HaNPV Polyhedrin Encryption Region An isolated DNA molecule containing the nucleotide sequence of a promoter. 31. Containing substantially all or part of the nucleotide sequence shown in Figure 1 An isolated DNA molecule characterized by: 32. Exogenous nucleotides located within the polyhedrin encoding and/or promoter region Recombinant HaNPVPH-, characterized in that it contains an otide sequence. 33. The exogenous nucleotide sequence encodes a substance harmful to insects. 33. The recombinant HaNPVPH- according to claim 32, having the following characteristics. 34. If the exogenous nucleotide sequence is of heterologous viral origin or or insecticidal compounds from scorpion venom, insect neurohormones, or Bacillus surin containing Bacillus thuringiensis δ-toxin. 34. The recombinant according to claim 33, characterized in that it encodes a substance selected from the group consisting of: Body HaNPVPH-. 35. The exogenous nucleotide sequence described above is functionally linked to the H8NPV polyhedrin promoter. 35. Any one of claims 32 to 34, characterized in that they are operably linked. The recombinant HaNPVPH- according to one of the above. 36. In a method for controlling the proliferation of pests in places where they grow, Insect virus according to any one of claims 1 to 4 or 32 to 35. or of occluded virus particles according to any one of claims 9 to 17. Including use in the above locations, the above insect viruses or occluded virus particles may be used in agriculture. A method as described above, characterized in that the admixture material is released together with a medium that is acceptable to the public. 37. HaNPVIE-1 encryption region and/or HaNPVIE-1 promoter An isolated DNA molecule characterized in that it comprises the nucleotide sequence of. 38. Containing substantially all or part of the nucleotide sequence shown in Figure 2 An isolated DNA molecule characterized by: 39. The gene or gene(s) that is normally responsible for stopping protein synthesis in the host A recombinant virus expression vector, which is inactivated.