JPH04501065A - Improved baculovirus expression vector - 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] Saletabakinirovirus Beta9- The United States Government has rights in this invention. This study was conducted by the National Institutes of Health Research funds N, R, H, No, Al23719 were supported.

1 spoon! The present invention relates to methods and compositions for improving gene expression. For more details , the present invention provides a new method for improving the expression of heterologous genes in baculovirus bases. Regarding regular promoters.

Soy bean paste Herein, the following definitions apply for the purposes of the invention.

The term "expression" can be characterized as follows. One cell contains many proteins. It has the ability to synthesize dark qualities. Many proteins that cells can synthesize are They are not always combined. a given polypep encoded by a given gene The gene is said to be expressed when Tide is synthesized by the cell. expressed In order for the DNA sequence encoding a given polypeptide to be Must be properly placed in relation to the area. The function of this control area is that The goal is to allow gene expression under the control of

The term "vector" refers to a double stranded vector containing a complete replicon that can be replicated in a cell. This refers to an extrachromosomal molecule that is the DNA of Vectors are generally viruses or bacteria. and from yeast plasmids. Bagyu 10 virus vector is Pacuroui Including Ruslepurifun.

The term "gene" means a protein that carries the information for the synthesis of a protein chain and that refers to the DNA sequence that directs the formation of

The term "infection" refers to an action on a cell under conditions favorable for cell replication and proliferation. This refers to the invasion caused by harmful substances (e.g. viruses, bacteria, etc.).

The term "transfection" refers to infecting cells with purified nucleic acid of a virus. It refers to the technology that makes

The term ``heterologous gene'' here refers to baquierovirus vectors, which are usually The vector is not produced by the virus from which it is derived, but as recombinant DNA. into cells, or reside within a virus containing a recombinant DNA genome. refers to the DNA that encodes the existing polypeptide. The term “parase” used here The term “heterologous gene” or “parasanger DNA” is synonymous with the term “foreign gene”. are equivalent. As used herein, "foreign" is synonymous with 'foreign'.

The term "transplacement plasmid" refers to the construction of viral vectors. refers to bacterial vectors used as intermediates in trans braces By homologous recombination, the endoblasmid transfers foreign genomic information to the viral genome. Promotes metastasis to specific areas. The above-mentioned exogenous genome information includes, for example, new promotional information. a combination of heterologous structural genes that exist under the control of a promoter and its promoter. It is set. This homologous recombination is caused by DNA sequences flanking the chimeric gene. arise.

Genetic engineering has reached the stage where certain biochemically useful products can be produced in large quantities. It has developed in For example, two commercially successful drugs, human growth hormone. protein and tissue plasminogen activator (t-PA) are now produced in large quantities. It is produced and used in various pathological treatments. However, scientists have New and more efficient ways to produce proteins and other products in chemical systems We are constantly trying to discover new systems.

The technology for transferring and expressing genes from one species to another is Chemically similar NAs with long chains containing the same four nucleotides This is possible because of the fact that The nucleotide sequence is basically Due to the same correspondence between amino acids and nucleotide sequences in all living species, It is arranged in codons (triblebuts) that code for amino acids. DNA is genetic Assembled into a gene containing control and food regions that mediate initiation of offspring expression It is.

These control regions are commonly referred to as 'promoters.'

An enzyme called RNA polymerase binds to the promoter region and activates it. be made or given commands in some way. This allows the fermentation The DNA moves along the coding region and extracts Messenger ribonucleic acid from the DNA. 2. Transfer the coded information to NA). The mRNA contains recognition signals. Ru. It provides signals for liposome binding and translation initiation and termination signals. It is a signal about null and boriadenylation. Next, cellular liposomes , the nucleotide codon information of +eRNA is determined by the nucleotide codon. It is translated into a protein with an amino acid sequence.

The general use of restriction endonucleases, as well as their ability to manipulate DNA sequences, , a double strand having the desired nucleotide sequence, containing the sequence of useful restriction sites. It became possible to chemically synthesize oligonucleotides with thicker (improved) has been done. Virtually any naturally occurring, cloned or genetically altered DNA segments that have been synthesized or chemically synthesized have the appropriate sequence or By connecting the recognition site to the end of the DNA molecule, any other segment Can be paired with both. This product can be produced by hydrolysis with a suitable restriction endonuclease. The purpose of this process is to generate the complementary ends necessary to join the DNA molecules. gene transfer Although there may be many variations of the scheme, the technique Appropriate fE placement and It is important to note that it is possible to insert DNA sequences in both directions. It is. Any DNA sequence is an artificial recombinant molecule, or a chimera, or is inserted into the vector molecule to construct a complex called hybrid DNA. There is a possibility that For most purposes, the vectors used are double-stranded. is an extrachromosomal DNA molecule that is transformed into a recombinant DNA molecule. Contains a complete replicon so that it can be replicated when placed in bacteria or yeast. have Commonly used vectors are viral or bacterial and It is a plasmid related to yeast.

Due to the nature of the genetic code, the inserted gene or portion of the gene is - binds to a control region (promoter) that can regulate expression in the cells in which it replicates If so, it directs the production of the amino acid sequence it encodes.

The cloned gene is placed in the proper relationship to the promoter region General techniques for the construction of expression vectors are described in the literature (T., Maniatis et al. (1982) Molecular Cloning A Laboratory Manual, Col1d Spring Harbor Laboratory ) is disclosed.

Many vector systems that utilize the general schemes and techniques described above are genetically modified. For use in the commercial or experimental synthesis of proteins by decorated organisms. It was developed for

In many of these vector systems, for vector replication and expression of heterologous genes, For this purpose, a prokaryotic 111rs host is used. Additionally, vector replication and heterologous Systems using eukaryotic cells for gene expression have also been used. Such a system Synthesis of hepatitis B virus surface antigen and human tissue plasminogen activity It is used in the synthesis of beta.

Eukaryotic hosts require modification after synthesis to become biologically active (e.g. glycosylation), desirable for eukaryotic protein production. Prokaryotic cells are one Generally, such modifications cannot be made.

The use of viral vectors in eukaryotic hosts has received a considerable amount of recent research. This is an issue in research. Viral vector systems reduce their usefulness There are serious disadvantages and limitations. For example, some viral vectors To economically produce proteins in an extremely expensive eukaryotic cell culture system , unable to reach sufficiently high levels of gene expression. in certain eukaryotic cells Viral vectors are pathogenic or carcinogenic in mammalian systems. , and the potential serious health and safety issues associated with outbreaks. There is a possibility that Some viral vectors contain There are severe limits on the size of a foreign gene that can be stably inserted into Ru.

As genetic engineering techniques become more sophisticated, more than one foreign gene, e.g. achieve the harmonious expression of genes encoding more than one protein, and The process of inserting gene products into host cells in order to obtain their harmonious activity. My interest is increasing.

The ideal viral vector stably stores large segments of foreign DNA. have the ability to infect a cell effectively, and have the ability to effectively infect a cell and Virtually all of the protein synthesis is altered to highly express the foreign gene. It has such abilities. of many heterologous genes in a highly eukaryotic environment, Viruses suitable as vectors for propagation and high-level expression are Irus AutOgrapha california nuclear polyhedrosis Keratinosis) virus (AeMNP■), (Miller, L., (198 1)-Virus Vector for Genetic Engineeri ng in Invertebrates, -Genetic Engines ring in the Plant 5 sciences, N, Panop olous ed., Praeger Publ., N. Y., pp. 203-224. ; U.S. Patent No. 4.745.051) The baculovirus group has The subgroups of drosisvirus (NPV) and granulosisvirus (ay) are exist.

Baculoviruses infect only arthropod hosts. Will of NPV and GV The particles are trapped in proteinaceous crystals. Baquillovirus and In the enclosed form, the pillion (enveloped nucleocapsi) ) is embedded in a crystalline protein matrix. This structure It is called a clathrate or a clathrate, but it is a form found outside the body in nature. Yes, and they play a role in spreading infection between organisms. Subgroup NPV is a large number of periods encapsulated in one large (up to 5μ) polyhedral crystal. produce. On the other hand, subgroup Gv consists of one pin encapsulated in a small crystal. Produce Lion. Crystalline protein matrix in either of its forms are known as polyhetrin or granulin in NPV or GV, respectively. consists essentially of a single 25kDa to 33kDa polypeptide It is.

More general information about the structure of baculovirus and the process of infection can be found below. This is in the review article. :　Carstens　(1980)-Baculoviru ses-Frfend or Man, Foe of Jnsects? , -T Rends and Biochemical 5science, 52:107 -10; Harrap and Payne (1979) - The 5tr uctural Properties and Indentificati on of 1nsect Viruses-in Advances in Virus Re5each4o1.25. Edited by M. A. Layer et al., Acad. e+sic Press, New York, pp. 273-355; and Ml 11er, L. (1981) (supra).

Baquinirovirus helper-independent viral vectors are especially biologically active. It is useful for the production of high levels of sexual eukaryotic proteins. (some foreign genes) The expression level is reported to be 10% to 25% of the total protein in recombinant infected cells. It has been tell. Single-chain peptide fragments, glycosylation, phosphorylation, polymerization, and complex forms Appropriate post-translational modifications such as synthesis, isolation and protein hydrolysis can improve this expression. A variety of atypical proteins produced using this system have been reported.

The baquinirovirus expression vectors disclosed to date are capable of directing foreign gene expression. For example, a very late promoter For example, using polyhetrin or polypeptide 10 (plo> promoter) There is. (Review by Luckow and Su+smerS (1988)-T rends in the Developments+ent of Baculov irus Expression Vectors, + old o/Technolo gy　6:47-55; Miller 几, Ni, (1988) -Baculo- viruses as Gene Expression Vectors,” Ann, Review of Microbiology, 42:177-19 9. ) These promoters are regulated during the process of viral infection and are very slow. It is activated during the infection process, which usually begins 18 to 24 hours after infection. Po The lehetrin and p10 genes are essential for replication in cell culture. It's not something. Therefore, this gene influences the production of virus in the budding state. can be replaced with the foreign gene in question without causing any problems. However, poly Hetrin gene replacement affects viral inclusion formation. recombinant plastic Regarding the recombinant virus, due to the absence of encapsulated virus in the Phenotypic selection can be made. This is useful, but somewhat complicated. this has also used recombinant technology to more cheaply produce large amounts of protein in insect larvae. Limits the possibilities of using viruses.

The economic value and general utility of baquinirovirus vectors is due to the It is highly dependent on the nature of the promoter used to direct expression. Polyhedo The phosphopromoter is widely chosen for high-level production of proteins. He is a promoter. However, often higher levels of protein production Required for economic viability.

In addition to high productivity, the vector must be capable of expressing one or more heterologous genes. It is considered essential. The disadvantage of such vectors is that the promoter arrangement within the vector is They are genomically unstable when the sequences are replicated. that anxiety To minimize stability, another promoter is used for each heterologous gene. It must be used for manifestation. These promoters are Naturally occurring viral promoters in the vector should be used to avoid instability issues. vectors should be non-homologous. Vectors can also be used for protein production. It must have the ability to contain a promoter that carries out gene expression in the early stage of the process.

This improves the quality of the protein and ensures that the protein undergoes any necessary post-translational modifications. It becomes biologically active or has immunological properties. This kind of translation Post-translational modifications can be made using the very late promoter of the AcMNPv vector, e.g. and plo are activated to decrease in the very late stages of infection. I can see it.

results in significantly increased expression of heterologous genes in the Baki10 virus system, Alternatively, it may be modified or synthesized to allow for the production of high quality proteins. A specific promoter region is required. The new promoter is fully adaptable A single heterologous gene, or a set of heterologous genes, can be used in a vector system. , thereby allowing various proteins to be released at once or at different times. can be produced. Additionally, if the vector can be administered orally to a suitable insect host, If so, the polyhetrin gene should be present.

Oitomo f1 piece The present invention places a heterologous gene under the control of a new or modified promoter. In order to express the heterologous gene by placing the baquinirovirus gene Improved methods of using expression vectors are provided. More preferably, the above promotion The promoter promotes high-level expression of genes and ensures proper production of gene products in insect cells. This is a novel promoter that enables post-translational modification. More specifically, preferred In embodiments, the present invention provides modified vakinirovirus with a heterologous gene attached thereto. This includes providing a promoter. The Promoter inserted into the virus by the smendo plasmid or by direct insertion, Recombinant viral vectors are produced that are used to infect appropriate host cells. Ru. The infected host cell is used to produce a heterologous gene product.

According to the present invention, the expression level of heteropolar genes or the collection of genes (college Methods and compositions for increasing the level of ctton of genes be disclosed. The present invention provides a natural B. Contains modified versions of virus promoters or synthetically modified promoters. It includes a new promoter. The new promoter has two start sites. A combination promoter such as two promoters (e.g. two ATAAG) or or a combination of promoters, such as two different promoter phase promoters. It can be a set. The present invention also provides a novel arrangement! or outside in the direction of the genome. It is also intended to encompass the location of future genes. All said promoters are listed here is referred to as a 'modified baculovirus promoter'.

The modified Babu Futsute virus promoter of the present invention has a region highly enriched in A4T. has one or more characteristics selected from the group consisting of ATAAG sequences with adjacent regions. are doing. This ATA, AC sequence is an activation sequence upstream of the transcription start site. It is located about 10 to about 30 bp upstream, and is a GC-rich sequence of about 10 bp. The linker scan promoters LSXVI, LSXIV, and L7 is similar to the upstream activating sequence of To prevent antisense transcription that occurs from 'rAAG sequences, is the polyadenylation site (A2[IA3 site) in the opposite configuration, and and one of the three very slow promoters (vp39.plO,pa,9) and/or very strongly expressed, very slow genes (polyhetrin, , pfo). The selection of these elements is rich presence in untranslated leader sequences of late and very late genes expressed in Based on conservation. An activator sequence upstream of the modified promoter 5 sequence) increases the transcription level at the transcription start site located downstream. It is an array that acts on

Baquinirovirus expression vectors use late or very late promoters. To be useful, the upstream active sequence must be a GC-rich sequence, with at least about 6 It has a nucleotide composition of 0% G+C.

The modified promoters of the present invention can be combined with other AcMNPV promoters to a limited extent. They simply have sequence homology or continuity. This new promoter The main advantage of the AcMNPv genome is that its AcMNP The point is that it can be stably integrated without causing recombination with other regions of the Y genome.

This modified promoter is designed to be stronger than the polyhedrin promoter. It can be done.

The modified promoters of the present invention can be modified by methods well known to those skilled in the art. It has been inserted into different trans-braced plasmids. For example, plastic psynVI has AcM between 3.1 and 6.16 map-L--/to In the NPV sequence, a synthetic protein replaces the EcoRV to Kpnl segment. Contains a motor. According to one aspect of the invention, the orientation of the promoter is in the direction of expression. direction is opposite to that of normal polyhetrin gene expression, and therefore the foreign gene When positioned in this orientation, it has the advantage of potentially increasing gene expression. outside The derived gene can be located in the multiple cloning site of this plasmid and the recombinant Viruses can be identified based on the encapsulated negative phenotype.

The second trans-braced plasmid psynVll-wtp is a synthetic protein. motor (with multiple cloning sites for parasenger gene insertion) ), and the polyhetrin gene under the control of the wild-type polyhetrin promoter. have This trans-braced plasmid is a polyhedrin-deficient mutant strain. When co-transfected with virus-derived DNA, polyhetrin and the foreign gene It is possible to construct (or form) a recombinant virus that directs the production of both products. Becomes Noh. The advantage is twofold. In other words, recombinant viruses can easily have a visible and readily selectable phenotype (positive enclosure), and The recombinant virus can infect insects orally. Therefore, it is easily found in insects. used for mass production.

Transbraced mendoblasmids contain a number of additions that increase expression in various conditions. can be manipulated in a conventional manner. For example, polyadenyls that are effective for inserting foreign genes Efficient polyadenylation occurs when no nylation sites are included. , at the multiple cloning site, and downstream of the foreign gene insertion. It is useful to include Gunar. For the construction of trans-braced blasmids The portion of the multiple cloning site that was not used was enriched in ACT near ATG. Baculovirus gene with highly expressed leader sequence and AACAAT sequence (i.e., vp39.plo, and pa,9). Since there is, it is deleted to make the expression ditto.

proteins modified from linker-scan modifications or from other promoter elements. The ability to construct promoters represents a novel challenge in designing promoters. It is only. Most of the components of the new promoter are in one theoretical order. Arranged in There appears to be flexibility in the order of elements in untranslated leader sequences.

Therefore, according to the present invention, a large number of heterologous genes can be synchronized by the modified promoter. expression by one vector.

One object of the present invention is that more than one heterologous gene can be expressed in one vector. We present a baquinirovirus vector with a modified promoter that allows It is about providing.

Yet another object of the present invention is the homologous recombination of a portion of the viral genome and its in the virus to virtually avoid the possibility of subsequent deletions or transformations. A modified promoter that differs sufficiently in its sequence from other promoters in The objective is to provide the following.

Yet another object of the invention is to provide a recombinant vector that can be orally administered to a suitable insect host. provides a quinyrovirus expression vector, resulting in an insect host virus vector system. It is possible to produce a desired irregularly shaped tanno chestnut material.

Yet another objective is to reduce the expression of heterologous proteins compared to polyhetrin systems. To provide a baculovirus-based expression vector that allows for the expansion of be.

These and other object features and advantages of the invention are described below in the disclosed embodiments. This is evident from the detailed description and appended claims.

A baculovirus capable of replication in cultured host cells is a vector of the present invention. useful for converting to Preferably, the baculovirus used is Nuclear polyhedrosis virus, more preferably Autographia It is californLea.

The vector of the present invention is prepared by genetic engineering techniques well known in the art, Preferably, the chimeric gene containing the modified promoter of the invention is inserted into It is prepared by Such insertion is the replication machine of the baculovirus mentioned above. homologous recombination in regions of the genome that can tolerate insertions without interfering with These can be done by performing the following steps, which will be obvious to those skilled in the art. The promo In combination with a foreign gene placed under the control of a baculovirus, It is prepared by inserting it into the nom.

Host cells useful for expression of heterologous genes under the control of the modified promoters of the invention As is well known to those skilled in the art, the term "vesicle" refers to the virus vector of the present invention, as is well known to those skilled in the art. is an insect cell capable of replication and expression, and is an insect cell cultured in vitro. Also includes insect cells and insect larvae.

The conserved sequences of the late and very late promoters of the invention are readily available to those skilled in the art. can be identified, and examples are given in Table 1.

Any upstream activation sequence known to those skilled in the art will enhance expression of the chimeric genes of the invention. used to. A preferred upstream activation sequence of the invention is a G of about tobp. +C-rich sequence, more preferably by the sequence CCAAGCTTGG. The old ndl+1 site linker is shown.

It is homologous to baculovirus, which acts as an expression vector, and the chimeric gene of the present invention Any trans-braces that flank the gene and have sequences well known to those skilled in the art. Blasmids may also be used in the practice of this invention. This homologous adjacent sequence is Chimeric gene homology from trans-braced mendoplasmids to loviruses It is understood that it must be long enough to cause recombination.

The trans-braced plasmids exemplified by the present invention are pi:v ss and its derivatives.

Brief description of the drawing FIG. 1 is a schematic diagram of a plasmid called PhcLSXIV.

FIG. 2 is a schematic diagram of a plasmid called psynV[-.

FIG. 3 is a schematic diagram of a plasmid called psynVI+matp.

FIG. 4 is a schematic diagram of a plasmid called I) SynwtVI-.

Figure 5 is a schematic diagram of a plasmid called psPLsXIVV[+cAT. .

FIG. 6 is a schematic diagram of a plasmid called pLsX[VVI+cAT.

FIG. 7 is a schematic diagram of plasmid phcvt.

FIG. 8 is a schematic diagram of plasmid pEV+eod.

FIG. 9 is a schematic diagram of plasmid pEV+aodX IV.

FIG. 10 is a schematic diagram of plasmid pSynV I-CAT.

FIG. 11 is a schematic diagram of plasmid pSynVI+vtpCATl.

FIG. 12 is a schematic diagram of plasmid psynwtYI-CATI.

FIG. 13 is a schematic diagram of plasmid pLsXIVsVI+.

FIG. 14 is a schematic diagram of plasmid pLsXIV2.

FIG. 15 is a schematic diagram of plasmid phc39.

FIG. 16 is a schematic diagram of plasmid pEVvp39/LSXIV CAT.

Figure 17 is an explanation of the plasmid schematic diagram.

A sad day According to the present invention, expression of genes in baquinirovirus expression vectors or Methods and configurations are provided for improving child collection. More specifically, the present invention is used to direct the expression of foreign genes and proteins produced during infection. We provide plasmids containing novel promoters for improving the quality of protein. Ru. This novel promoter can be synthesized from synthetic promoters, natural promoters, etc., depending on your needs. modified promoters and/or natural, modified or synthetic promoters. may contain combinations. This new promoter also supports the combined promotion Contains a meter. It has two initiation sites (i.e. two TAAGs). or, for example, early/very late promoters, or late promoters. / of two different baculovirus promoters, such as the super-late promoter. including but not limited to combinations.

The present invention also provides the ability to It is intended to include the placement of foreign genes.

Insect Baki xo virus Autographa caI fornica to nuclear poly Drosis virus (AcMNPV) is widely used as a gene expression vector. (Review by Luckov and Summers, Trends in the Development of Baculovirus Expressi on Vectors.

B1o/Technology 6:47-55, 1988; Miller, L , , Baculovirusesas Gene Expression Vectors, Ann, Review of Microbiology.

42:17? -199, 1988). Helper-independent viral vector systems are Generally, the very late viral promoter, the polyhedrin promoter or Foreign genes expressed in the AcMNPV genome under the control of the plO promoter Includes insert. These two novel promoters support high levels of each gene. transcription takes place, so that mRNA It is believed to be particularly useful as it provides high steady state levels of A. Enclosure , i.e., the virus particles are embedded in a pseudocrystalline protein matrix. Despite being required for effective oral infection of host larvae, cell culture It is not essential for the growth of viruses in nutrition.

Therefore, it is important to relocate specific genes to the inclusion stage and to replace the inclusion genes. Expression of foreign genes at high levels is a cause of infection during cell culture. Does not have any observable effect on production by non-encapsulated viruses (budding viruses). Don't lose it.

The baculovirus expression system is based on the structure of the baculovirus gene or Initially, based on very little knowledge of the properties of viral promoters, developed. (Miller, L., '1., 1981) Polyhedrimp First trans bracemen developed for vector systems based on promoters One of the doblasmids was pAc373. This is the normal level of gene expression Previously it was reported that some of the sequences required for the vector were not present in this vector. It was widely used (MatSuura et al. (198))-Bacul.

Virus expression vectors: The required ments for high 1 level expression of p protein, 1ncludiB glycoproteins-J.

Gen, Virol, 68:1233-1250) o Polyhetrin gene +1 (A of ATG) containing the nucleotide immediately upstream of the position Ismendo plasmids are now recommended for vector use (Luck, supra). ov and Summers (198g); Miller, L., (198 g)).

+1 downstream (e.g. within the polyhetrin open reading frame) The nucleotide at the position is not required for optimal transcriptional efficacy (Carb onell et al. (19Bg)"Synthesis of a gene encoding an insect-specific scorpion neurotoxin and an attempt to express it using baculovirus” Gene, ?3:4 09-418).

Linker scan of the region immediately upstream of polyhetrin encoding sequence according to the invention Mutational analysis revealed several new properties of this region. promoter The first determinant of activity is with respect to the ATG (+1.+2.43) of the translation initiation site. , -50 in the sequence TAAGTATT of the transcription start site. transcription opening Sequences connected to the start site can be used to control the level of transcription as well as reporting by more sophisticated methods. It affects the expression of target genes. 10 to 30 upstream of TAAGTATT Rearrangement of the linker in the sequence increases transcription levels and gene expression by 50%. make it big On the other hand, TAAGTATT and +1 position (i.e. untranslated lead) Relocating the linker between the sequences that specify the programmer region The motor activity decreases by 2-fold to 10-fold regulation. reporter gene and 93 this gene Polyhetrin "promoter" consisting of cleotide (i.e. +1 to -9 Observation in the natural direction by reversing the genome direction of (up to 2) Expression of the reporter gene is at the same or slightly higher level than when Become a le. None of the mutations alter the temporal regulation of gene expression. qualified All gene expression from the polyhedrin promoter is restricted to very late stages. (Rankin (198g) “Baquinirovirus polyhedrin gene expression” 8 base pairs surrounding the transcription start site and key determinants for Gene 7 0:39-49. References incorporated herein).

The very slow polyhetrin and plO promoters are probably responsible for foreign gene expression. Despite being considered the most desirable promoter for The relative ability of slow promoters to direct gene expression has not been directly demonstrated. There is no. most encoding vp6.9 (core) and vp39 (capsid) Two rapidly expressed slow viral structural genes have been located and sequenced. were determined, and a transcriptional map was determined (Wilson et al. (1987) “Small Location and location of baculovirus encoding a small arginine-rich polypeptide Transcription and sequence "J, Virology, 61:661-666). 12 hours postinfection and before transcription from the strong polyF-phosphorylated promoter. The image was fully transferred in about 12 hours. The products of the vp6.9 and vp39 genes are Required for encapsulated and budding forms of the virus. Therefore, these manifestations During the very late period after infection, polyhetrin is present for both continuing and foreign genes. p10 promoter is expected to take a longer time than the p10 promoter.

, specific expression of a foreign gene product at a very late stage indicates that the product is considered desirable for the expression of foreign genes that interfere with sexual viral synthesis. (Miiler, L, K.

(1981)).

However, many foreign gene products do not adversely affect virus synthesis; , as well as earlier and higher levels of expression are advantageous from a production perspective. slow Expression in stages.

It is also more preferred for effective post-transcriptional modification at very late stages. host nucleus m RNA transcripts decrease dramatically between 12 and 24 hours post-infection. and Post-translational modifications are first conveyed by the host gene product, but appears inefficient at a very late stage. The inventors have demonstrated that slow capsid promotion The vp39 promoter promotes the expression of foreign genes at a late stage. I found it to be very good at guiding people. The polyhedrin promoter is activated after infection. Performance increases approximately 5 to 8 times between 12 and 24 hours.

The present invention uses non-viral polyhetrin, such as the natural viral polyhetrin as well as the p10 promoter. Enables higher level expression of foreign genes than would be produced by a consistently slow promoter. This includes improvements to pachinirovirus gene expression vectors to produce a pachinirovirus gene expression vector. present invention also contains two different research methods = (1) polyhedrin promoter modification of upstream sequences connected to the transcription start site, and (2) to direct foreign gene expression. slow vp39 (capsid) promoter or slow/very slow promoter This is the use of a combination of meters. Reverse the genomic arrangement of the promoter and foreign gene appears to have little effect on the level of gene expression, and Two or more viruses directed by one Bakibistovirus vector allows the design of plasmids for simultaneous transfer and expression of incoming genes.

In addition, according to the present invention, the novel synthetic promoter It was designed to have minimal DNA sequence homology with the promoter.

Such promoters can be used to generate more than one foreign promoter using one recombinant virus. Useful for expression of genes as well as natural viral promoters in vectors – Eliminating the need to duplicate arrays. Thereby, repeated promoter sequences Potential instability of the genome due to homotypic recombination between columns is minimized.

As part of this invention, we facilitated the formation of baquinirovirus expression vectors capable of: discloses a trans-braced plasmid that is mediated by a trans-braced plasmid. (1) Gene (2) simultaneous expression of two parasenger genes; (3) increased levels of expression; ) Parasenger gene expression at very late and late stages, and / or (4) identical genes for both the parasanger gene and the polyhetoone protein gene. Time expression. Transbraced mened plasmids are useful for foreign gene insertion Restriction sites, i.e. multiclonins at appropriate sites downstream of the promoter by providing a genetic site and allowing the foreign gene to be perfected. By connecting the viral DNA sequences from the region of the virus genome, Facilitates insertion of foreign genes into the nirovirus genome. Will connected The virus sequence contains both non-recombinant viral DNA and recombinant viral vectors. between the recombinant trans-braced mendo plasmid and the cells in order to provides a site for more mediated homologous recombination.

The inventors have demonstrated that linker-scanning mutations upstream of polyhetrin TAAGTATT are compared to the wild-type polyhedrin promoter (Rankin (1988) supra). The researchers found that the protein increased transcription and foreign gene expression. As shown in Figure 1 Plasmid phcLsXIV.

and has the same promoter and basic multiple cloning site but C pEV odXIV lacking the AT gene is a linker-scan mutant promoter It is currently called the LSXIV promoter. peEV＋＊od XIV-derived viruses or pheLSXIV-derived viruses are known Results in higher levels of reporter gene (CAT) expression than Carscan modification . A virus carrying the CAT reporter gene under the control of the LSXIV promoter Expression from the polyhedrin promoter (i.e., ph containing the wild-type polyhedrin promoter shown in Figure 10, derived from cvt. (compared to CAT gene expression from a recombinant virus). This was shown to be 50% higher than the current level.

Therefore, preferred embodiments of the invention provide for various upstream sequences of the TAAGTATT sequence. Some plasmids contain promoters with modifications of . 1st A plasmid called phcLsXIV shown in the figure has such a modification. This is an example of a plasmid containing a promoter. Other programs with this property The lasmids include phcLsXV I and phcLsXYII (Table 5). When these plasmids are integrated into recombinant viruses, Contains an upstream modified promoter that results in high level expression of the gene.

In another preferred embodiment, the present invention comprises a synthetic plasmid. It has a plasmid. Synthetic promoters are used to direct heterologous gene expression. It provides diversity in the nucleotide sequence of promoters and enhances the expression of foreign genes. Bringing differentness to the levels. Sequences for preferred synthetic promoters are shown in Table 1. Well illustrated by the DNA sequence shown. The arrow in the DNA sequence indicates the start Indicates point and direction of transfer.

It is flexible to allow for the arrangement and ordering of the elements of a promoter, and is shown in Figure 2. It is understood that the sequence of its promoter shown is an example of an embodiment of the invention. be done.

A trans brace member of the second embodiment containing the synthetic promoter described above. As an example of a doblasmid, its general construction is shown in Figure 2. psynV This plasmid, designated I-, carries the synthetic promoter shown in Table 1 and here A transbreeder with a multiple cloning site (MCS) called MCS#2. It's Ismendo Blasmid. The MOS$2 sequence of psynVI- is shown in Table 2. ing. The orientation of the synthetic promoter allows foreign gene expression to occur in the opposite direction of the genome. This is what happens when the orientation is . Construction of this promoter was performed using wild-type polyhedrin Directs expression of the heterologous gene at approximately 10% to 20% of the level of the promoter.

Another example of the second preferred embodiment of the invention is pSynV I+vt It is a plasmid called p. A diagram of this promoter is shown in FIG.

This construction, similar to pSyn'/I- described above, uses the synthetic promoter described above and MCS #2, and is a transfer vector containing the wild-type polyhedrin promoter. Under its control, the polyhetrin gene is expressed. This means that two genes are expressed simultaneously. This is an example of a plasmid that can be used. This plasmid has a selectable encapsulation phenotype. Since the recombinant virus is encapsulated, insects can be infected orally. It can be used for. The construction of this plasmid is based on the native polyhedrin promoter. Leads to approximately 10% to 20% heterologous gene expression.

Another plus included as an example of the second preferred embodiment of the present invention Mido is psynvtV! -, which is shown in FIG.

This is a polyhedrin promoter (with an MCS site, MCS #1) and A transcript containing an adult promoter (with a second MCS site, MCS#2) It is a sbraid plasmid and is characterized by the expression of two foreign genes. There is. This trans-braced plasmid therefore contains two genes. This is an example of an expression vector. However, other synthetic promoters Place the polyhedrin promoter based on the principles demonstrated for the promoter. Uses the polyhedrin promoter because it can be designed to change It is considered that there is no need.

In a third preferred embodiment of the invention, the plasmid is Contains a promoter or several promoters with an orientation opposite to that of do. psPLsXIVVI+CAT and pLsXIVVI+cAT (5th The plasmids shown in Figures and Figure 6) carry the pLsXIV promoter in the opposite orientation. (with respect to the original polyhetrin orientation). these pluses mid to test CAT expression from the LSXIV promoter in the reverse orientation. used for. The pLsXIVVI+cAT plasmid is a tandem double He is a promoter. CAT gene from LSIIV promoter in reverse orientation It is true that offspring expression can be comparable to the level achieved in the original orientation. discovered by the inventor.

The promoter may be placed in different genomic locations/orientations to increase the level of expression. It is contemplated as part of this invention that it may be located in any gender. Plasmid psPL sXIVVI+cAT (Figure 5) and pLsXIV3VI (Figure 13) are two examples of high level expression vectors in opposite orientations.

A fourth preferred embodiment of the invention is the vp39 (capsid) promoter. Includes plasmids containing the sequences. The DNA sequence of this promoter is shown in Table 3. show. Transbrace membranes that can be used to test reporter-CAT expression The endoplasmids include phc39 and pEVlod39. that vp3 9 promoter, compared to the natural polyhedrin promoter, at 12:00 p.i. This results in approximately 5- to 120-fold higher levels of expression during the 24-hour period. Also a book The invention can, for example, improve early gene expression as well as higher finality of heterologous proteins. levels (total levels) such as polyhetrin and vp39 promoters. It is intended to encompass such slow/very slow promoter combinations. infection Because it is expressed earlier in the process (i.e., in the case of polyhetrin, 1 postinfection) (showing up after 6 hours rather than 8 hours), the slow promoter? High level expression is obtained by 12 hours after infection. slow/very slow promotion The combination of tars should ideally be activated at about 6 hours post-infection and subsequently at 70 hours post-infection. It continues to operate over time, thus resulting in slow and very slow gene expression.

Mu Promoter and A Promoter Lance Srace Mendbrass Mido! The inventors have demonstrated that an important determinant for polyhetrin gene expression is ATAAGT It was discovered that it appears to be located in the ATT sequence.

Transcription of abundantly transcribed slow genes is associated with ATs connected by ACT-rich regions. A novel bacteriophage that occurs in the AAG sequence (Wtlson et al., 1987, supra). The rus promoter is shared with other abundantly expressed slow and very slow genes to other viral promoters by joining a short sequence motif of and are designed to lack minimal sequence homology.

Two additional features incorporated into the design of the new promoter are ATAAG with the LSIIV linker for the upstream G+C-rich region and near the ATG. This is the TTTATT sequence located in the untranslated region. The TTTATT is a foreign gene insertion. Polymerization for transcripts that occur in the opposite orientation from the ATAAG sequence, which happens to occur during Serves as an adenylation signal.

The inventors have determined that slow and very slow promoters are the most common transcription start sites. It was discovered that ATAAG is contained in .

This sequence is important for polyhetrin expression as well as promoter activity. moreover , linker-scanning mutational analysis of the polyhedrin promoter – nucleotides appear to contribute to optimal gene expression throughout the region. It showed. Promoters of the four most frequently expressed genes of AcMNPV The sequence characterizing the untranslated leader region of the main capsid protein is slow gene encoding basic core protein (p6.9); a slow gene encoding plO, a very slow gene encoding plO, and a polyhetri These genes are arranged in a - column. All four areas are rich in ACT. Contains ATAAG.

Several short segments found in two or more promoters has been selected as a component of synthetic promoters. 2 or more The longest common segment of promoters is 9 nucleotides TAAAT TACA, found in both pIO and p6.9 promoters . ACAAT sequences are present in 3 of the 4 promoters (not polyhetrin). , is expressed near ATG, and TACTGT is expressed in polyhetrin and p10 promoters. TTTGTA was discovered in both plO and brihedrin promoters. and TCAANTCA was found in the pIO and p39 promoters. difference In addition, all promoters have a stretch of at least three T's, Most are connected at A and have a stretch of at least 3A. , usually preceded by a pyrimidine. All these components are synthetic It was designed as a promoter model. This component is designed to minimize length. For example, the T stretch and A stretch are located immediately downstream of ATAAG. It acts as an ACT-rich part downstream of ATTAC.

However, the ACAAT sequence links the promoter to the multiple cloning site. close to the matching 3' restriction site (BglTI).

and TTTATT (which in retrograde is the polyadenylation signal) , is connected to this sequence and from ATAAG in a manner that promotes termination of the reverse transcript. located as far away as possible to avoid interference with the antisense RNA at the 5' end. Minimize harm. Syn protein synthesized to construct the synthetic promoter model Two oligonucleotide sequences, called romators, are labeled with component markings. are shown in Table 1.

The trans-braced mendoplasmid pSyn- (Figure 2) is a polyhedrin protein. Contains the Syn promoter that drives expression in the reverse direction as a motor. That's it In addition, the multiple cloning site (MCS#2) has a large number of useful restriction sites. ) is also included. In addition, the 627 nucleotide encoding the N-terminus of polyhetrin Therefore, I decided to use this trans brace mend blasmid. This results in a recombinant with an inclusion-type negative phenotype.

The synthetic promoter and associated multiple cloning site (MCS#2) Restriction endonuclease (e.g. downstream Sac of MCS#2 or promoter) to other AcMNPV genomic locations using pn+ or Sea lig upstream of the target. can be moved to other plasmids for transfer.

The invention is further illustrated by the following examples. This makes the present invention in no way There are no forced restrictions. On the other hand, when reading this description, the invention as suggested to one skilled in the art without departing from the spirit and/or scope of the claims. It is understood that the present method encompasses various other embodiments, modifications, and equivalents thereof. can be clearly understood.

(Margin below) Trap Wataru plate Example 1 All viruses are originally derived from AcMNPVL-1 and are derived from Lee and Mi. 11e’r (197B) “A, cal 1fornfca nuclear polyhedrosis Isolation of Genotypic Variants of the Virus"J, Viro1.27:754-767) , plaque method aughn et al. (1977) - Insect 5podoptera fr 2 from ugiperda (Lepidoptera: Noctuidae) Establishment of two insect cell lines” In Vi Koushi 13: 213-217, previously The method reported in Lee and Miller (1978): Mille (1986) ``Insect baculovirus for high-level expression of foreign genes'' host vector system “genetic engineering, principles res, N, Y, pp, 2 77-298.1986).

According to previously reported methods (Carbonell et al. (1985)'' Bacterial genes in Diptera insect and mammalian cells are mediated by Bakico and Loviruses. J, Vjrol, 56:153-160HRankin et al. (1 9138) supra:) CAT assay was performed and specific activity was calculated. child CAT genetics for all CAT-containing plasmids used in these studies. The offspring are pcMlcAT (LK B Biotechnology, Piscataway, LJ. ) due to come

Example 2 pEViodXIV) The construction of the lance bracemend plasmid (Figure 9) It will be revealed. This served as the basis for the construction of the aforementioned transfer plasmid phcvt (Fig. 7), It also serves as a starting plasmid for constructing pEV+modX IV. pEY55 The lasmid contains 3.18 to 7.3 ma-nobu units (lU) of AcMNPvDNA. Contains. The multiple cloning site co-locates sequences from +1 to +635. Replaces polyhetrin, which is coded. pEV5snoptlc hector 1 site and 3, Many restriction endonucleos at the Sal 1 site connection of 18 AcMNPV DNA. The crease site determines the type of restriction site that can be used in the multiple cloning site. do.

Therefore, this connection site is removed by pEV55, which is digested by SmaI. removed. This is to cut at the vector connection point, as well as the Exor The combined action of nuclease and mung bean nuclease generates approximately 70 nucleases at the connection point. It is removed by deleting the cleotide. Ligation at this deletion site to obtain plasmid pEVdel. 6.16wu and 7.3 in pEVdel Viral sequences between +ou and Ban old (6°16 mu) and Notl (7,3+ou). deleted pEVmod (Fig. 8) is obtained by blunt-end ligation of the connecting point C. . The polyhedrin promoter of pE'/sod (-92 EcoRV to +IB gllll) is a small EcoRV-Bgl of phcLSXIV (Fig. 1) l! Convenient Blasmitote A6 pEV that has been replaced and improved with segments Obtain modX IV. Trans brace mendob based on pEVmodXIV Viral vectors derived from rasmids can be pEV55 or any other known vector. produces approximately 50% higher levels of expression than plasmid-based viral vectors .

Example 3 phc39 (Figure 15) and I) E'Vt1lod39 (Figure 9) trans The construction of a sbraithmend blasmid is described. Contains the vp39 promoter A plasmid is first constructed as follows. 57. on the AcMNPV genome. Nar I to gcoR containing the vp39 promoter between 6 and 57.9 mu The V segment was constructed using the plasmid vector Bluescript KS-(Strata gene, San Diego, CA), both /1 ccl and EeoRV. It is cloned to the position. This resulting plasmid, psTVNM, was 1 and the Apai site. The terminal sequence of the sequence encoding vp39 is , excised by exonuclease III and mung bean nuclease digestion. removed. Bgll+linker oligonucleotide is made using DNA ligase. inserted into the gap. - compared to vp39's ATG (+1.+2°+3) One plasmid containing the Bgl11 site at 2 is selected. p39pr For the construction of Kpn! Oyo and EcoRV from the previously selected plasmid, and Kp Plasmid Bluescript Kst (Stra O trans brace connected to ngene, San Diego, CA) For construction of the endoplasmid phc39, p 39pro's 458bp segment from EcoRV to Bgllll was deleted, followed by EcoFIV and Bgl I! to phcvt digested with inserted. Thus, the polyhedrin promoter of phc sulfate is the vp39 promoter. Can be replaced in the motor area. The trans brace mendo bra obtained in this way Sumid phc39 (Figure 15) was used to construct the viral vector vhc39. It will be done. CAT using this vector between 12 and 24 hours after infection. Gene expression is 5 to 10 times higher than that of CAT driven by the polyhedrin promoter. The level will be twice as high. Earlier expression of foreign genes results in more efficient protein production. resulting in effective post-translational modifications. More preferred trans brace mend brass Mid, e.g. pEV+*od39 (not shown), Bgl11 and and a Kpnl terminus (e.g. MCS #1). It can be easily obtained from phc39 by hatching.

Example 4 At the position of the polyhedrin promoter, there is a Syn promoter and an adjacent multi Plasmid pSynVI- containing the cloning site (MCS#2) (Fig. ) to construct the two Syn promoter oligonucleotides shown in Table 1. The two strands are annealed and the two strands are purified by polyacrylamide electrophoresis. Bluescript plasmid was prepared and then digested with Kpnl and EcoRV. vector pBSKS (Stratagene, San Diego, C, A , ) is inserted into. This Bluescript plasmid can be used for multiple cloning The site lacks the Sma I restriction enzyme site. This allows BlueScript Plasmi to Forced re-cloning of oligonucleotides into the multiple cloning site of Also, it becomes possible to reproduce both EcoRV and Kpnl.

The multiple cloning site of this plasmid spans the 5scl site from EeoRV. The parts include EcoRV, EcoRI, P+st I, old Bag, and 5pel , Xbal, Notl, 5acll and 5scl sites in this order. Le. Its synthetic promoter and this multiple cloning site are linked to 5scl and man. Blue script is digested in an orderly manner by Gumbean nuclease and Xpnl. vector. The small segment is gel purified and then pEVmod digested with Kpnr and EcoRV and gel purified (Figure 8) inserted into the larger segment. From S+ga 1 site called MCS#2 , the promoter sequence via adjacent multiple cloning sites is determined.

The sequence is shown in Table 2. The 5scl site was unexpectedly added during the cloning process. It is played back accidentally.

The action of mung bean nuclease downstream of the 5scl site is responsible for the observed ligation. becomes.

The psynV I plasmid containing the reporter-CAT gene was constructed psynVI by measuring CAT gene expression on AT-containing plasmids. A plasmid is constructed to measure the level of gene expression.

In the construction of the psynVI-CAT plasmid (Figure 10), the CAT gene was , deleted from pcMlcAT by 5ail and cleared by mung bean nuclease. It is made blunt-ended and inserted into psynVI which is cut by EeoRV. C.A.T. At the N-terminal part of the gene, directionality was confirmed by using the EcoR1 site. Ru. The corresponding viral vector vsynV I-CAT is a polyhedrin promoter. levels observed with viral vectors expressing CAT under the control of It results in CAT expression at a level of 10% to about 20%. polyhedrin promotion This expression from a synthetic promoter is constitutive, although not to the same extent as tar expression. Level is important. Syn promoter and the resulting promoter The motor carries out specific post-translational modifications of two different foreign genes and/or It is useful for the expression of genes encoding regulatory proteins.

Example 5 Enables foreign gene expression and polyhetrin gene expression in some applications Carrying the genetic information to form a recombinant virus is a transbreeding process. Useful for ismendoblasmids. Expression of both genes promotes inclusion body formation. Providing efficient oral infection of insect larvae for cheap and large-scale protein production possible. Furthermore, production in insect larvae is carried out in differentiated insect cells (the insect's main (including fat bodies that constitute secretory tissues), such as SF9 cells. more efficient post-translational modifications than seen in useful lepidopteran insect cell systems. bring.

Therefore, psynVI (Fig. 2) is a trans-braced plasmid p modified to produce synVI+ matp (Figure 3). This is polyheto It may be possible to allow both Lin and the foreign gene to be expressed at high levels at the same time. p Another advantage of synVl+vtp is that the polyhetrin-deficient mutant (or Polyhetrin/β-galactosidase fusion mutant) DNA is a viral D Co-introduces early into host cells with NA and trans-braced plasmids. When transfected, the recombinant virus is present as inclusion body-positive plaques. It is about having the ability to choose.

The psyr+vI+matp trans-braced plasmid is an AcMNPV gene. A plasmid carrying the EcoRI-1 segment of Nome was transformed into EcoRV and Kpn. The polyhedrin promoter and N-terminal polyhedrin region were digested using Constructed by deleting . EcoRV/Kpnl segment of approximately 0.7kb The sample was gel purified and psynVl digested with SmaI and Xpnl. − will be inserted. Smal and EcoRV will be deleted by Ligege Seal. However, Kpnl found in the polyhedrin hood region is regenerated. More details contains the polyhedrin promoter and EcoRV (-92> to Kpnl ( The N-terminal coding sequence up to Inserted into psy+1VI- to produce ynVI+tp. in this way , polyhetrin + mRNA is normally transferred to something under the control of a normal promoter. The foreign gene is cloned into a multiple cloning site under a synthetic promoter. inserted and transcribed in the opposite direction relative to polyhetrin. the plasmid The characteristics are shown in Figure 3. EcoRV/S+*al connection and synthetic promoter The sequences of the vector and multiple cloning site (MCS$2) are shown in Table 1.

To test the effectiveness of the transfer vector psynvl+vtp, reporter-C The AT gene is EcoR'/site "t" psynVI+vtp (Figure 3). Inserted into MC5, plasmid psynVI+vtpccATI (Figure 11) is obtained. For construction of psynV I+CAT1, pCM-1CAT ( 1) The blunt-ended CAT-containing 5ail segment was digested with EcoRV. pSynVI+vtp. Simultaneously with polyhetrin gene expression, The ability of Syr+ promoter to drive the porter-CAT gene, recombinant virus vsynVI+vtpcAT1 was isolated, and the level of CAT expression was determined by vsynVI+vtpcAT1. Tested by comparing with the level obtained by VI-CAT. this No significant differences in CAT gene expression were observed between the two viral vectors. First, we show that co-expression of the polyhetrin gene does not interfere with foreign gene expression. Ma Furthermore, the recombinant virus vSynVI+vtpCATl produced high levels of inclusion bodies. Therefore, the polyhetrin gene is abundantly expressed.

Example 6 Trans brace men blasmid psynv t V! - construction (Figure 4) explained. This plasmid has two promoters in close proximity; Simultaneous transfer of two heterologous genes and cooperative expression of two genes by this vector is possible. The pEV55EcoRV-Kpnl segment is a polyhedrin romator and multi-cloning site MCS$1, Small and inserted into psynVI digested with Kpnl.

psynvtVI- containing the reporter-CAT gene is psynvtVI- constructed to serve as an indicator of gene expression. The plasmid is psynvtV I-CA, TI (Figure 12). Construction of psynvtVI-CATI Mainly, the blunt-ended CAT-containing 5a11 segment of pCM-1cAT Inserted into EcoRV digested psynvtV+-. Equal amount of viral vector vs. ynwtV1-CATl is a CAT expression vector under the control of the polyhevilin promoter. It expresses CAT activity at 10% to 20% of the level observed in the microorganisms.

Example 7 pSPLSX IVVI+CAT and pLsXIVYI+-CAT trans The construction of bracemend plasmids (Figures 5 and 6) is illustrated.

For the construction of psPLsXIVVl+cAT, the CAT gene and the connected The LSXJY promoter was digested with Xpnl (as well as mung bean nuclease). ) and phcLsXIV (more blunt-ended) by EcoRV digestion. 1) to pSynVI+vtp using EC0RV digestion. inserted. This places the LSXIV promoter in tandem with the synthetic promoter. Place the tar. The plasmid was then cut with EcoRV and Ipnl. and large vector segments are gel purified. Wild type polyhedrinpro AcMNPV Ec, oRI-1 segment containing the motor and end The EcoRV/Kpnl segment was gel purified and pSPLSXIVVt +CAT vector segment (EcoRV/Kpnl) By using the CAT gene under the control of the LSXIV promoter, a polyhydrin protein was generated. pLsXIVVr+cA containing the complete polyhydrin gene under motor control produces T. These Plax F (vSPLSXIVVI+CAT and vL sXIVV + cAT) derived +7) viral vector is a wild type polyhedrin Under the control of promoters, CAT can be produced in equal amounts compared to viruses that express CAT. Rui is expressed at slightly higher levels. Virus derived from pLsXIVVI+cAT Yet another advantage of this vector is that it produces high levels of polyhetrin. That is.

Example 8 pLsXIV3VI+ and pLsXIV2) Lance brace mend blasmi The construction of the board (Figures 13 and 14) is explained. Plasmid pLsXIVV I+cAT is digested by Bgll and EcoRT, and the sticky ends are extracted from mung bean. removed by nuclease. The DNA is cut by Small and a large section The fragment was gel purified and then carried with EcoRV and Sac ends. pL linked to the multiple cloning site (MCS$3) and lacking the CAT gene form sXIV3VI+.

This plasmid is then digested with EcoRV and Kpnl.

The large segment was isolated and pEVmodXIV (OEcoRV/Kpn 1) is inserted into the ``large segment''. two neighbors each with its own MCS pLSX IV2 has the LSXIV promoter. 9LSXIV3V The l+ plasmid uses the strong LSIIV promoter to express foreign genes. can be achieved at levels comparable to polyhetrin gene expression. pLsXIV2 The lasmid contains the top of two foreign genes, each under the control of the LSIIV promoter. enable expression.

Example 9 The trans brace mend plasmid phe39 (Fig. 15) is the vp39 pro. Constructed by a CAT reporter gene under the control of a motor. vhc39 Expression of CAT from vhcvt at 12 hours post-infection.

The CAT gene of phc39 is replaced with the foreign gene in question. Feeling Expression earlier in the staining process allows for more effective post-translational modification of heterologous protein products. is possible.

Example 1O Transbraced mendoblasmids insert two foreign genes into a viral vector. The recombinant virus allows for the simultaneous transfer of both foreign genes. Constructed to allow abundant expression of children simultaneously. transbrai The smendo plasmid psynvtVI (Fig. 4) has such a dual gene site. This is an example of a lance braced blasmid.

This plasmid can be used to digest psynVI- with Smal and Kpnl. and pE containing wild-type polyhedrin promoter and MC11. Constructed by inserting the V5sEeoRV/Ipn+ segment. death This plasmid therefore contains two different multiple cloning sites, downstream of the adjacent Syn and wild-type polyhedrin promoters, respectively.

Example 11 Another example that allows for equally high levels of a second gene is pLsXIV2 ( Fig. 14). This plasmid carries the flanking LSIIV promoter. each of which contains restriction sites useful for inserting two different foreign genes. has multiple cloning sites (MCS#l and MCS#2), each of which It is under the control of the LSIIV promoter. The efficiency of the adjacent promoter is vLs Tested using XIVVI+cAT. This is the polyhedrin promoter A recombinant with pLsXIVVI+cAT$if construction adjacent to r　CAT expression This virus causes polyhetrin gene expression. vLsXIVVI+ cAT hec9-Niyor CAT expression is caused only by phcwt, which only expresses CAT. comparable or slightly higher levels compared to when observed.

Example 12 A vp39/polyhedrin promoter fusion construction is described.

The Bindlll linker is inserted into the 462 bp promoter of vp39.

The DNA sequence of vp39 is shown in Table 3 (see Rankin at al. (1988) supra, for a general linker scanning approach).

Bindlll linker creates slow/very slow combinatorial promoters In order to A combinatorial promoter that acts to allow 988) +1 of polyhedrin promoter of LSIIV promoter (above) vp39 site fused to approximately -60 (e.g. -65 to -350)17) Constructed to contain regions of OCT-A and OCT-B. To do this In order to This plasmid is selected (e.g. vp39-65). From Hindll The segment to Bg’llll is Hindll in phcLSXIV. The segment from ll to Bgl[I is replaced. Combination body vp39/L The entire segment from Bglll to EcoRV containing the SXIV promoter ] is transferred to P.EVS5, forming a trans-braced plasmid, i.e. pEVvp39/LSXIV with a combination promoter controlling CAT expression Construct CAT (Figure 16).

Example 13 Trans-braced plasmid pEVvp39/LSXIV (Figure 16) The construction of was accomplished according to the method described in Example 12 above. hybrid v The p39/LSXIV promoter sequence is shown in Table 4. vp39/LSXIV group The connection point sequence of the combined promoter is Htndl of the connection point (CCAAGCTTG). 11 sites and approximately 1 to 10 nucleotides of vp39 at the junction (Nl-1 0) is marked by "N1-10 CCAAGCTTG".

Of course, what has been described above pertains to preferred embodiments of the invention. , and any additional claims hereafter, which depart from the spirit and scope of the present invention. It should be understood that many modifications or alterations may be made here. Must be.

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Claims (1)

[Claims] 1. Baquiulovirus vectors for producing gene products in host cells - the vector comprises a modified baculovirus promoter and a heterologous gene, the heterologous gene being under the regulatory control of a modified promoter. It is in. 2. The modified baculovirus promoter is activated by the modified promoter. has a polyadenylation site in the direction opposite to the direction of transcription that is directed, and 2. The adenylation site according to claim 1, wherein the adenylation site is located 3' to the transcription start site. vector. 3. The modified baculovirus promoter contains two initiation sites. , the vector according to claim 1. 4. The modified baculovirus promoter is a late promoter of baculovirus. 2. The vector according to claim 1, which has a conserved DNA sequence of /very late promoter. Tar. 5. About 1 of the GC-rich sequences located about 10 to about 30 bp upstream of the transcription start site has an upstream activation sequence containing 0 bp, and the upstream activation sequence comprises the modified promoter. 2. The vector of claim 1, which increases expression directed by a vector. 6. The modified baculovirus promoter is phcLSXIV, phc A plasmid selected from the group of plasmids consisting of LSXVII and phcLSXVI. 2. The vector of claim 1, which is isolated from Sumid. 7. The vector according to claim 1, wherein the novel promoter comprises the DNA sequence shown in Table 1. ctor. 8. The modified promoter is the modified late promoter of pEVmod39. The vector according to claim 4. 9. The modified pSPLSXIVVI+CAT promoter is 2. The vector of claim 1, which is isolated from the midge. 10. Plasmid pSynVI-, pSynVI+wtp, pSynwtVI- , pLSXIVVI+, and pLSXIV2 modified baculovirus proteins. having a baculovirus promoter selected from the group consisting of promoters; The vector according to claim 1. 11. 2. The strain according to claim 1 for the production of multiple gene products in a host cell. A eurovirus vector comprising multiple modified baculovirus promoters. - and a vector comprising the heterologous gene of claim 1. 12. The baculovirus expression vector is derived from nuclear polyhedrosis virus. 13. The vector according to claim 1. The nuclear polyhedrosis virus is Aut ographica californica virus or a mutant of c. 13. The vector according to claim 12. 14. Baculovirus expression vectors suitable for expression of heterologous genes in host cells This is a method for preparing baculovirus that has been modified to the baculovirus genome. including the insertion of a virus promoter and a heterologous gene; is under the regulatory control of the modified baculovirus promoter. 15. The baculovirus expression vector is a baculovirus that replicates in insect cells. A virus and a transplacement plasmid are contacted, and the foreign gene is Selecting a recombinant virus that expresses the gene product encoded by the offspring. 15. The method of claim 14, formed by: 16. Claim 14, wherein the Maeda baquidurovirus is a nuclear polyhedrosis virus. Method described. 17. The nuclear polyhedrosis virus is Autoraphica califo 17. The method according to claim 16, wherein the virus is a Nica virus or a mutant thereof. 18. A regulated baculovirus promoter having the DNA sequence of Table 1. 19. The modified Baki contained in plasmid pSPLSXIVVI+CAT Urovirus promoter.