KR890002414B1 - Method for producing a recombinant baculovirus expression vector - Google Patents

Abstract

The method of preparing expression vector of recombinant vaculovirus comprises: (a) cloning DNA fragment containing AcMNPV polyhedrin gene into EcoRI of plasmid PUC8; (b) extracting AcNMPVDNA from virus and separating by cesium chloride equilibrium density gradient centrifugation; (d) treating AcMNPV DNA with EcoRI restriction endonuclease; (e) cloning AcMNPV-EcoRI-I fragment into pBR 325 and subcloning to form pI8; (d) seperating plasmid losing PstI, BamHI, SmaHI and EcoRI.

Description

Method for producing a recombinant baculovirus expression vector

FIG. 1 is a schematic diagram of the process of manufacturing the transition vector pAc101.

2 is a schematic diagram of the fabrication process of modified transition vectors pAc311, pAc360, pAC373 and pAc380.

Figure 3 schematically shows the partial nucleotide sequence of the polyhedrin gene of AcMNPV and the immediately upper sequence of that gene.

4 is a schematic diagram of a method for cloning an IFN-β gene into a transition vector to produce a recombinant expression vector pAc 380-IFN-β.

FIG. 5 transfects the recombinant expression vector pAc 380-INF-β with baculovirus in cultured Spodoptera frugiperda cells, and cultures the cultured S. pruperfera cells. It is a schematic diagram showing the process of continuous infection with plaque purified recombinant baculovirus.

Figure 6 shows the recombinant transfer vector pAc 380-IFN-β inserting the IFN-β gene into the BamHI cloning site of the EcoRI fragment of the AcMNPV genome.

The present invention relates to a method for producing a recombinant viral expression vector. More particularly, the present invention relates to a method for fusing a selection gene coupled with a baculovirus promoter into the baculovirus genome to produce a recombinant baculovirus expression vector capable of expressing a selection gene in insect cells.

Recent advances in recombinant DNA technology have been used to isolate specific genetic residues or portions thereof and transfer them to viruses, yeast, plant or animal cells and viruses that infect these organisms. The transferred genetic material (or modified gene) is replicated and multiplied by the replication of the transformed cell or virus. In turn, the transformed cells gain the ability to produce products encoded by the transcribed gene sequence. The DNA of all organisms is chemically similar in that they consist of four identical nucleotides, allowing the transfer and expression of genes or parts of them between viruses, prokaryotes and eukaryotes. The basic difference is the nucleotide sequence that appears in the organism's genome. Specific nucleotide sequences arranged in codons (nucleotide triplets) encode specific amino acid sequences. However, the coding relationship between amino acid sequences and DNA nucleotide sequences is essentially the same in all organisms.

Genomic DNA consists of protein coding sequences (ie "structural genes") and regulatory sites that regulate the expression of structural genes (a DNA sequence that regulates transcriptional initiation is referred to as a "promoter"). In general, RNA polymerase is transcribed into mRNA as it is activated by a promoter and moves along structural genes. mRNA contains recognition sequences, signals for ribosomal binding, and signals for translation initiation and termination. Recent advances in genetic analysis of the role of important transcriptional signals in the promoter region of genes (generally described as the 5 'flank of a gene) have led to selective DNA sequences to study the role and role of DNA sequences in expression. The possibility of removing or modifying and the possibility of removing specific DNA sequences has emerged to study the function of DNA sequences in heterogeneous biological systems such as recombinant DNA host-vector systems. Eukaryotic promoters are generally characterized by two conserved nucleotide sequences whose position and structure are similar to prokaryotic promoter sequences, suggesting that they are involved in transcriptional promotion (Breathanch & Chambon, 50 Ann. Rev. Biochem 3490383 ( 1981).

The first is an aninine and thymine-rich sequence in the nucleic acid (Goldberg Hogness, "TATA" or "ATA" box), 20-30 base pairs located at the top of the RNA initiation (cap region, the mRNA transcription initiation), and the consensus sequence. It is specified as (5'-TATAA-ATA-3 '). The second region is the CCAAT box (Efskatadis, et al 21 Cell 653-668 (1980)), which uses the 70-90 base pairs located above the cap region of several genes to express the standard sequence 5'-GG (C / T) CAATCT-3 '. (Benoist, et al, 8 Nucleic Acids Res 127-142 (1980)).

Such sequences may also be removed and modified with restriction endonucleases to cause controlled removal or modification of the nucleotide sequences that have been cloned to produce recombinant DNA molecules and cloned by in vitro or specific site mutations. Restriction endonucleases are hydrolases that can catalyze the cleavage of specific regions of DNA molecules. The active site of a restriction enzyme is determined by the presence of a specific nucleotide sequence, which is a recognition site by the corresponding restriction endonuclease. Many restriction enzymes have been isolated and classified according to their recognition sites. Some of these restriction endonucleases hydrolyze phospho-diester bonds at the same position on both DNA strands to create blunt ends, and other endonucleases contain several nucleotides from each other. Hydrolysis of the bonds separated by means yields a single chain portion in a free state at each molecular end. The single chain ends can be used to recombine complementary single-chain sequences that are identical to autologous and hydrolyzed DNA or heterologous DNA sequences. Restrictions are relatively rare. However, the general use of restriction endonucleases has been greatly improved by using chemically synthesized double stranded oligonucleotides containing the desired restriction site sequences. Substantially any naturally occurring cloned, genetically altered or chemically synthesized DNA fragment can be paired with any other cross section by binding an oligonucleotide containing an appropriate recognition site to the end of the DNA molecule. The product is subjected to the hydrolytic action of a suitable restriction endonuclease to provide the complementary ends necessary for pairing with DNA molecules.

The recognition sites for specific restriction enzymes are usually irregularly distributed. Therefore, cleavage by restriction enzymes occurs within codons or between adjacent codons at some arbitrary site of the gene. Many different changes are possible to this summary, but to express these sequences, it is important to acquire useful techniques for inserting DNA sequences in the proper position and orientation relative to the promoter site.

Provisionally, any DNA sequence may be cloned by inserting a heterologous DNA sequence into a cloning carrier or vector molecule to construct a complex called an artificial recombinant molecule or chimeric or hybrid gene. For most purposes, the cloning medium used is an extrachromosomal double DNA sequence containing complete replicons so that the recombinant molecule can be replicated when placed by transformation into bacteria, yeast, plant or animal cells. to be.

Commonly used cloning media are viruses and plasmids bound to bacteria, yeast, plant and animal cells.

Recent advances in biochemistry and recombinant DNA technology have made it possible to produce cloning media or vectors containing "heterologous" DNA. The term “heterologous” means DNA encoding a polypeptide that is not normally produced by a cell. Heterologous DNA may be maintained in the cell either fused into the genome of the cell or transformed with a self-replicating plasmid or virus cloning medium. The transformed cell population provides a source of regeneration of heterologous DNA for other manipulations, modifications and transfer to other vectors. Certain viral vectors carrying heterologous genes will replicate in the transformed cells and lyse the transformed cells. During replication, the heterologous gene may or may not be expressed within that particular cell type. Cloning viruses can be isolated and used to infect other cells and provide a source of regeneration for recombination for other uses.

The gene or its preferred portion has been cloned and / or biochemically modified in the required way or other biochemically modified gene or genomic gene has been inserted in such a way as to facilitate its expression and they are transferred to the expression vector. do. Because of the nature of the genetic code, cloned or hybridized genes or parts thereof induce the production of amino acid sequences that the gene encodes. General methods for constructing expression vectors with cloned genes for unique relationships with promoter sites are described in B. polisky, et al., 73 Proc. Natl. Acad. Sci. U.S.A. 3900 (1976), K. Itakura, et al., 198 Science 1056-1063 (1977), L. Villa-Komaroff, et al., 75 Proc. Natl.Acad.Sci. U.S.A.3727-3731 (1978) and the like.

The term "expression" can be characterized in the following method. Even in relatively simple prokaryotic organisms, cells have the ability to synthesize many proteins. At some point, the cells do not synthesize as many proteins as they can synthesize. When a particular polypeptide encoded by a given gene is being synthesized by a cell, that gene is said to be expressed.

In order to be expressed, the DNA sequence encoding the particular polypeptide must be appropriately located relative to the control region of the gene. The function of regulatory sites is to allow the expression of genes under the control of regulatory sites to respond to the cell's changing needs at any given point.

The following definitions are used throughout this specification: Cloning media are extrachromosomal double DNAs containing complete lipolycons that can be replicated by transformation in cells or organisms. In general, cloning media are derived from viruses or plasmids. Most take the form of circular DNA.

The term gene refers to the DNA sequence involved in the synthesis and delivery of a single protein chain.

The term infection refers to invasion by pathogenic viruses in cells that have good conditions for their replication and growth.

The term transfection refers to infecting a cell by purifying the nucleic acid by DNA precipitation and adding calcium chloride to the DNA solution containing a suitable agent such as phosphate or dextran sulfate to absorb it into the cell. It means to make a technique.

Many host-vector systems used in the general overview and techniques described above have been developed for use in commercial or experimental protein synthesis by genetically modified organisms. Most of these host-vector systems are eukaryotic host-vector systems as described in US Pat. No. 4,338,397 to Gilbert et al. In addition, A. Miyanohara et al., 80 Proc. Natl. Acad. Sci. U. S.A. Systems used for yeast as a vector, such as systems used for surface antigen synthesis of hepatitis B as described in 1 (1983) and systems for human interferon synthesis in yeast as described in 219 Science 620 (1983) by Pitzeman et al. It has been.

The value of prokaryotic host vector systems for desirable protein synthesis is reduced due to the specific restriction enzymes inherent in such systems. As an example, mRNA transcripts or protein products of the system may be unstable in prokaryotes. In addition, DNA sequences into which microorganisms are introduced prior to protein synthesis in prokaryotic cells include intervening DNA sequences that encode polypeptide sequences that do not contain active eukaryotic proteins, nonsense sequences, and initiation or terminal sequences. There should be no. In addition, some eukaryotic proteins require modification in order to be biologically active after synthesis, and prokaryotic cells generally do not have such capacity.

Other limiting factors associated with yeast or prokaryotic host-vector systems include difficulties associated with recovering the synthesized gene product from within the cell. US Pat. No. 4,336,336 to Silhavy et al. Details the problem of recovery of gene products and provides a method for synthesizing and secreting proteins by genetically modified bacteria.

The use of viruses in advanced host-vector systems has been the subject of a significant amount of recent research and consideration. However, viral vector systems also have significant disadvantages and limitations that reduce their usefulness. For example, many eukaryotic viral vectors are tumorigenic in mammalian systems, thus creating the potential for serious health and safety problems associated with the resulting gene products and accidental infections. In addition, in some eukaryotic host virus vector systems, the gene product itself exhibits antiviral action, which reduces its protein production. 1 J. Molec. Applied Genet. By D. Gheysen and W. Fiers. In the eukaryotic host virus vector system described in 385-394 (1982), the yield of 40 of Simian monkey viruses caused by only 100 units of interferon is reduced by 80%. Another inherent problem with the use of eukaryotic host-virus vector systems lies in the form of viruses. For example, viruses have very few restriction sites, making it easy to insert foreign DNA into simple viruses at specific locations. However, eukaryotic genes are too large to be inserted into simple viruses.

Thus, the shape of the virus limits the amount of foreign DNA that can be packed into a simple virus. On the other hand, since complex viruses having many restriction sites have many restriction sites, it is more difficult to insert foreign DNA into the complex virus at a specific position.

The present invention overcomes many of the limitations described above by using promoters in the baculovirus and the baculovirus genome to produce viral expression vectors in eukaryotic host-vector systems. More specifically, the baculovirus Autographa californica (AcMNPV) and the polyhedrin promoter contained therein produce recombinant viral expression vectors capable of expressing selected genes at very high levels in eukaryotic host insect cells. It has been found that it can be used to The resulting gene products of this system can be sufficiently secreted into cell media and alleviate most of the difficulties associated with the recovery of protein products. Also more importantly, it is not tumorigenic in mammals of this system. The theoretical advantages associated with the use of baculovirus in the eukaryotic host-virus vector system are described in more detail by L.K. Milley's "A Virus Vector for Genetic Engineering in Invertebrates". (Panopoulos, N.J. (Ed.), Genetic Engieering in the Plant Sciences (New York, Praeger Publishers, 1981), pp. 203-224.

In a broad scope, the present invention provides a method for producing a viral transfer vector, a recombinant viral transfer vector and a recombinant viral transfer vector. The obtained recombinant viral expression vector can express a selection gene in a host insect cell.

According to the present invention, a baculovirus virus comprising a baculovirus gene or a part thereof containing a promoter of a baculovirus gene is cleaved to obtain a DNA fragment (powder) containing at least the promoter. In a preferred method, a DNA comprising a flanking DNA sequence and a polyhedrin gene of a suitable baculovirus, such as the preferred baculovirus autographa californica (AcMNPV), is first isolated. The required DNA is cleaved by suitable restriction enzyme methods. It produces a DNA fragment comprising a polyhedrin promoter and at least one DNA sequence encoding a polyhedrin protein or part thereof. One such DNA fragment is an EcoRI-I fragment comprising a DNA sequence encoding a polyhedrin promoter and a polyhedrin protein.

Next, the DNA fragment described above is inserted into a suitable cloning medium such as plasmid pUC8 to prepare a transition vector. Thus, a preferred transition vector, termed a polyhedrin transition vector, consists of a suitable cloning carrier comprising a polyhedrin promoter and a site useful for cloning the selection gene or a portion thereof such that the selected gene is under display control of the polyhedrin promoter. . Preferred transition vectors may or may not include DNA sequences encoding polyhedral proteins or portions thereof.

The recombinant transfer vector is then prepared by inserting the selected gene or portion thereof into a useful cloning site of the transfer vector described above. Probably any gene can be cloned into the transition vector of the invention and paired with the baculovirus promoter sequence. In addition, by appropriate recombinant DNA techniques, the DNA sequence encoding the polyhedrin protein can be deleted from the preferred transition vectors described above so that the resulting cloned gene product is the protein of choice alone. Otherwise, if the coding sequence for the polyhedrin protein is not deleted or if at least one coding sequence for the polyhedrin protein is not deleted from the desired transition vector, the resulting cloned gene product is selected protein and polyhedrin protein or portion thereof. Hybrids containing will also be fusion proteins.

To prepare a recombinant expression vector, the recombinant transfection vector is contacted with the appropriate baculovirus DNA to induce recombination and thereby fuse the necessary genetic material into the baculovirus genome. A preferred method of completing recombination is known methods of transfection. Since the transfection process does not occur 100% for the virus, the result is a mixture of non-recombinant and recombinant. Recombinant baculovirus expression vectors capable of expressing a selection gene in host insect cells are then selected by appropriate selection or genetic selection techniques from recombinant and nonrecombinant baculovirus mixtures. One method of selecting an expression vector is achieved by identifying and separating viruses lacking viral uptake in the nucleus of infected cells by insertion inactivation of the polyhedrin gene.

The present invention also provides a recombinant baculovirus expression vector produced by the method described above. The expression vector includes an infectious baculovirus comprising at least one selection gene or portion thereof that is stable within and is integrated with the viral genome. While an expression vector is replicated in an insect cell or insect, the selection gene may be expressed under the control of a baculovirus transcriptional signal or the control of its own promoter. A typical baculovirus expression vector is a recombinant AcMNPV virus containing a gene of β-interferon inserted at a position in AcMNPV under the electronic regulation of the polyhedrin promoter. There is the possibility that any baculovirus promoter and insect cell line can be used.

The present invention also makes it possible to produce transition vectors by the method described above. Preferred polyhedrin transition vectors include cloning sites useful for insertion of the polyhedrin promoter sequence and the selection gene or portions thereof to allow the selection gene to be under transcriptional control of the polyhedrin promoter and to be used as an intermediate medium for the genetic manipulation of baculoviruses. do. The polyhedrin transfer vector does not contain any or all of the DNA sequences encoding the polyhedrin protein.

The present invention also provides a recombinant transfer vector prepared by the method described above. Preferred recombinant transfer vectors of the present invention comprise a selection gene or part thereof coupled with a polyhedrin promoter and are used as a medium to fuse necessary genetic information into the baculovirus genome.

The present invention describes the use of the baculovirus promoter in a host-vector system to promote expression of select genes in eukaryotic insect cells. In particular, because polyhedrin proteins are one of the most abundantly synthesized proteins in virus-infected eukaryotic host cells, the preferred method is to fuse the selection genes into the appropriate baculovirus genome so that the selection genes are paired with the polyhedrin promoter. will be. Such recombinant baculoviruses provide an effective mechanism for the synthesis of select gene products. Thus, the present invention has significant utility as required gene products, such as β-interferon, are synthesized and effectively isolated from host insect cells at very high levels.

1 is a schematic diagram of a method for producing a transition vector pAc 101 starting from the flag purified strain AcMNPV, M3 plasmid pBR 325 and plasmid pUC8.

2 is a schematic diagram of a method for constructing modified transition vectors pAc311, pAc360, pAc373 and pAc380 starting from plasmids pI10, pUc8 and a synthetic BamHI linker. “Library” herein refers to a library of modified pB'Bal plasmids constructed by inducing deletion mutations at each possible position in the polyhedrin gene. Plasmids pB'Bal 11, pB'Bal 60, pB'Bal 73 and pB'Bal 180 are selected from the above library of mutant plasmids to change back to the transition vectors pAc311, pAc360, pAc373 and pAc380.

3 shows a partial nucleotide sequence of the polyhedrin gene of AcMNPV and the sequence immediately above it. In addition to the position of the only BamHI cloning site, the positions where deletion mutations are induced to construct the transition vectors pAc101, pAc311, pAc360, pAc373 and pAc380 are indicated by arrows. The "TATA" and "CAAT" boxes of the polyhedrin gene are indicated by rectangles drawn around the sequence and the transcriptional start site of the gene is indicated by an asterisk. 3 also shows EcoRV and HindIII restriction sites located near the polyhedrin gene.

4 shows a schematic of cloning a preferred IFN-β gene into the transition vector pAc380 to produce a recombinant expression vector pAc-IFN-β. 4 also shows starting material plasmid pBR13 comprising the IFN-β gene. Plasmid p770 contains a 767 base pair HincII fragment conjugated by pUC8 sequence with a synthetic octanucleotide BamHI linker. The 767 base pair fragment comprises the entire coding sequence of IFN-β.

FIG. 5 shows the recombinant baculoviruses transfected with baculovirus in cultured Spodoptera frugiperda cells and the cultured S. pruperfera cells were plaque-purified. Shows the schematic of continuous infection with Rovirus.

6 shows the recombinant transfer vector pAc380-IFN-β having the IFN-β gene inserted at the BamHI cloning site in the EcoRI fragment of the AcMNPV genome. The polyhedrin promoter sequence is shown in bold black and the EcoRI-I sequence is shown fused into plasmid pUC8.

The baculovirus used in the practice of the present invention is Autographa californica (AcMNPV). This baculovirus is characterized as follows.

Spontaneously infectious AcMNPV is commonly found as a virus bite. This virus aggregate generally contains several virus particles embedded in a para-crystalline protein matrix containing subunit structural arrangements of polyhedrin proteins. The occlusal body is ingested by a suitable host insect and when it reaches the intestinal lumen, occlusal separation occurs in the alkaline state to release the virus.

Viruses invade cells of the barrier and migrate to the nucleus of those cells to replicate. Two forms of infection are produced intracellularly, i.e., an occlusal virus and an extracellular or compared virus. The cell's virus germinates from its cell surface and infects other cells. About 12 hours after infection, cell germination decreases, polyhedrin synthesis starts, and the production of occlusal virus particles is increased. A large number of occlusal bodies are formed in infected cells and tissues, resulting in lysis of insects. This occlusal form of virus can spread infection to other insects.

Viruses of cells that are readily cultured in cell culture media are used in the typical methods of the present invention. Extracellular and occlusal viruses have the same genome but feature different phenotypes.

Polyhedrin, the major structural component of the occlusion, is a protein of about 29,000 molecular weights. The characteristic of the AcMNPV genome is that the gene for the AcMNPV polyhedrin is arranged about 4000 base pairs from zero to the base of the DNA restriction enzyme map for adjacent DNA sequences of about 1200 base pairs in the EcoRI-I fragment (see GESmith, JM). Vlak and MDSummers, 45 J. Virol. 215-225 (1983)).

A map of the complete AcMNPV genome is presented by J.M.Vlak and G.E.Smith. 1118-1121 (1982), and the DNA sequence for the polyhedrin gene is found in 131 Virology 561-565 (1983) by Hooft van Iddekinge, G.E.Smith, and M.D.Summers.

The structure and action of polyhedrin proteins are of considerable interest because they are one of the most highly expressed eukaryotic genes known. Polyhedrins accumulate at high levels in Spodofterapruperferda cells infected with AcMNPV, ie, accounting for more than 50% of the total amount of protein in infected cells, resulting in more than 0.2 grams of polyhedrin protein per liter of infected cells. It is. The gene is also noted for the following reasons: (1) The polyhedrin gene of AcMNP includes a DNA sequence that is highly conserved among baculoviruses. (2) recombination between closely related strains occurs at high frequencies at this site of the genome, thus allowing the blocking and expression of polyhedrin genes in the recombinant, and (3) gene expression to host, tissue and cell lines. It depends. From a genetic engineering standpoint, this polyhedrin gene is unnecessary because the virus can be replicated in the cell culture without the polyhedrin gene. Due to the high expression of this gene and the fact that it is not essential for viral replication, the possibility of using the polyhedrin promoter and the AcMNPV gene as part of a system for the expression of recombinant genes has been the subject of considerable research. (See EBCarstens, STTjia W. Doerfler, 99 Virology 386-398 (1979); P. Dobos MACochran, 103 Virology 446-464 (1980); HA Wood, 102 Virology 21-27 (1980); JE Maruniak MD Summers, 109 Virology 25-34 (1981); LKMiller, "A Virus Vector for Genetic Engineering in Invertebrates," In: NJPanopoulos (Ed.), Genetic Engineering in the plant Sciences (New York, Praeger Publishers, 1981), pp.203-224; LKMiller, et al., 219 Science 715-721 (1983); GESmith, MJ Fraser, MD Summers, J. Virol 584-593 (1983). Could not.

Experiments by Applicants have shown that other gene products up to 10K are also expressed in high concentrations comparable to polyhedrins (GESmith, JMValk and MDSummers, 45 J. Virol215-225 (1983)). It is obviously unstructured, produced late in infection, and produced in large quantities. The position of the 10K AcMNPV protein gene is located for HindIII fragments P and Q. Like the polyhedrin promoter and the structural gene, this promoter has been the subject of research for its advantageous use as part of a system that uses AcMNPV for the expression of recombinant genes. However, prior to the present invention, no system has been developed that allows for the advantageous use of such promoters.

By the illustrated method of the present invention, a specific monarch of AcMNPV, M3 or E2 is used. However, one of ordinary skill in the art having the benefit of the present invention will appreciate that other baculoviruses and other baculovirus strains may be suitable for producing recombinant baculovirus transmission and expression vectors. In particular, 30 J. Virol. 828-838 (1979) and GE / Smith and MD by closely related and naturally occurring baculovirus strains Tricho-Plusiani MNPV, Rachiplusia ou MNPV, Galleria mellonella MNPV and GESmith and MDSummers Plaque-purified strains such as the E2, R9, S1 and S3 strains of AcMNPV described and characterized by 33 J. Virol, 311-319 (1980) by Summers can be advantageously used. A more detailed description of this and other strains is described in 89 Virology 517-527 (1987) by G.E.Smith and M.D.Summers.

Advantageously used by the methods of the invention are polyhedrin structural genes and promoters. The gene was mapped by S1 nuclease experiment. The nucleotide sequence at the 5 'end of the polyhedrin coding site and the 200 bases on the top of transcription start are shown in FIG. The sites indicated in FIG. 3 are transcription start sites for the most frequently used polyhedrin mRNA.

The ATG translational start signal (the A residue at +1) occurs at about 58 bases from the start of transcription, followed by open reading up to 255 bases, including the HindIII site. The "TATA" and "CAAT" boxes found at similar positions in many eukaryotic genes are located between the top 25 and 35 bases and between the 60 and 70 bases, respectively, from the site of transcription. At the base centers of 78 and 90 at the top from the transcription start site is the repeated sequence "CACAACT". In addition, the AcMNPV polyhedrin gene has an untranslated leader sequence of 58 bases located in front of the translational codon, and there is no interference sequence as suggested by the appropriate experimental procedures for AcMNPV polyhedrin mRNA. See Smith, Valk and Summers, G. F. Rohrman, et al., 121 Virology 51-60 (1982).

In the practice of the present invention, DNA having a polyhedrin gene is isolated and purified from Baculovirus AcMNPV. Those skilled in the art will recognize that it is very beneficial to possess the polyhedrin gene and to be able to isolate the gene from other baculoviruses, particularly the related strains described above.

The DNA of interest is a 7.3 kilobase EcoRI-I fragment or other suitable fragment comprising a polyhedrin gene, which is degraded into EcoRI restriction endonuclease by appropriate restriction enzyme methods to prepare it.

The EcoRI-I fragment described above is cloned into the EcoRI site of a suitable cloning carrier to prepare a transition vector. Since AcMNPV does not have a unique restriction site to be known in which the selection gene is effectively inserted in a site-specific manner, it is necessary to construct chimeric plasmid vectors (transition vectors) that act as intermediate carriers for gene transfer. Therefore, in order to fuse a selection gene into the viral genome adjacent to the polyhedrin promoter sequence, a transition vector designated as the polyhedrin transition vector is constructed, which comprises the polyhedrin gene, the cloning site and the lateral viral DNA. The cloning site is characterized in that the gene appropriately inserted into this site is under the control of the polyhedrin promoter, and the lateral viral DNA is bound to one of both sides of the polyhedrin gene. The fabrication of the transition vector is shown schematically in FIGS. 1 and 2. In addition, flanking viral DNA presence promotes recombination with the wild form of baculovirus and allows the transfer of selected genes into the cloned viral genome.

Thus, the EcoRI-I fragments described above are cloned and subcloned into plasmids pBR325 and pUC8, respectively. Two BamHI Restrictions in an EcoRI-I Fragment to Create a Polyhedrin Transition Vector Named pAc101 and Having a Single BamHI Cloning Site Located 3 'Direction Above the Polyhedrin Promoter Sequence of About 220 Bases from the Translational Initiation of the Polyhedrin Gene The site can be removed (see Figure 3). Although plasmids pBR325 and pUC8 are plasmids used for the preparation of polyhedrin transition vectors, other suitable cloning carriers can be used provided that the polyhedrin gene and the lateral viral DNA are functionally fused.

The polyhedrin transition vector may be modified to insert a selection gene by deleting some or all of the coding sequence of polyhedrin synthesis near the end of the transcriptional start or polyhedrin gene (see Figure 3). DNA linkers containing natural or synthetic oligonucleotides comprising the BamHI restriction site sequence are inserted at the deletion site to enable pairing of the DNA fragments at that restriction site. Modification of the transition vector is shown schematically in FIG. 2, and the deletion of the polyhedrin coding sequence uses in vitro mutation. However, other methods may be used to delete some or all of the polyhedrin coding sequence, other synthetic or native oligonucleotide linker sequences may be inserted at the deletion site, and other modifications may be fused to the selection gene or portions thereof. It is a fact recognized by those skilled in the art that a polyhedrin transition vector can be suitably used in the present invention.

Selected genes, such as the IFN-β gene encoding human β-interferon synthesis, the CAT gene encoding chloramphenicol acetyltransferase synthesis and the IL2 gene encoding human interleukin-2 synthesis by standard cloning techniques, produce recombinant transfer vectors. To be inserted into the polyhedrin transition vector at a useful restriction site. Insertion of the β-interferon gene into the transition vector pAc380 is shown in FIG.

In addition, IFN-β, CAT, and interleukin-2 are typical genes cloned into polyhedrin transition vectors and paired with polyhedrin promoter sequences, but any selection gene can be used in the present invention, or in the modifications described above. You will notice that there is.

The hybrid polyhedrin-selecting gene, a recombinant transfection vector gene, is transferred into the genome of a suitable baculovirus, such as baculovirus AcMNPV, to produce a recombinant viral expression vector capable of expressing a gene encoding β-interferon in host insect cells. The transfer of the hybrid gene is carried out by the method of transfection in host insect cells such as Spodoptera pruperferda. J. P. Burand, er al. 101 Virology 286-290 (1980). The method is shown schematically in FIG. 5 using the recombinant transfer vector pAc380-IFN-β. After transfection, during replication of the AcMNPV DNA, the hybrid gene is transferred to AcMNPV DNA by recombination between the recombinant transfer vector and the AeMNPV DNA. Thus a mixture containing non-recombinant and recombinant baculoviruses is prepared and the recombinant baculoviruses can express the IFN-β gene. Although transfection is a preferred method for transferring hybrid genes into the baculovirus genome, other methods may be suitable for inserting hybrid genes into the baculovirus genome. In addition, recombination can be performed between the recombinant transfer vector and other strains of baculoviruses, as long as there is sufficient homology between the hybrid gene sequence and the corresponding sequence of the other strain for recombination to occur. For example, the recombination occurs between genes isolated from any of the strains Trichoplusia ni MNPV, Rachiplusia ou MNPV and Galleria mellonella MNPV and AcMNPV strains E2, R9, S1 and S3 described above. Recombination may occur at sites in the genome that do not explicitly contain homologous sequences. This mechanism is not explained.

Recombinant AcMNPV expression vectors comprising hybrid polyhedrin-selection genes, genes fused into the AcMNPV genome, are selected from non-recombinant and recombinant baculovirus mixtures. One of the methods of selection is to select plaques formed by infected host insect cells with a virus (named 0 ⁻ ) that does not produce virus bite. Selection is facilitated in this way because recombinant viruses are defective in the production of virus bite due to insertional inactivation of the polyhedrin gene. An average of 0.5% of virus plaques made from progeny virus 6 in transformed cells are made from putative recombinant 0 ^- virus. Thus, the DNA of the 0 ^- plaque forming recombinant virus is purified and digested with a suitable restriction enzyme to confirm that the recombinant AcMNPV vector inserts the select gene into the appropriate EcoRI-I position.

While the selection process described above provides an effective and conventional method of selecting recombinant baculovirus-selective gene expression vectors, other selection methods utilized by the present invention may be used. A relatively conventional method of detecting in situ heterologous DNA in eukaryotic cells (i.e., hybridizing labeled DNA probes to viral DNA present in plaques made from infected animal cells) has been described by Villarreal and Berg in 196 Science 183-186. 1977) and 15Gene 53-65 (1981) by Hayday, et al.

Expression of the selection gene is performed by infecting susceptible host insect cells, such as Spodoptera pruperferda, with a recombinant baculovirus-selective gene expression vector in a suitable growth medium. AcMNV expression vectors are propagated by replicating in insect cells or insects and assembling into infectious virus particles. The infectious AcMNPV expression vector may also be used to prepare a selection gene in suitable insect cells and thus promote sufficient expression of the selected DNA sequence in infected cells.

During infection, an AcMNPV expression vector-specific mRNA complementary to the DNA sequence of the select gene is produced. Vector-specific mRNAs are generally translated into infected cells to produce proteins that are fully or partially encoded by the select gene, and in some cases the select gene product is glycosylation, secretion, phosphorylation, or other post-translational modification. The same process as for post-translational modification.

Whether the gene product produced by the recombinant AcMNPV expression vector consists of the amino acid sequence of the protein of choice alone or is a hybrid protein comprising one or more additional amino acid residues derived from the amino terminal end of the AcMNPV polyhedrin or a protein of choice and hybrid protein. Whether to produce both products depends on how the polyhedrin transition vector is modified. If a single translational start signal (ATG) derived from a selection gene is present in the hybrid gene sequence and the selection gene is present in the hybrid gene sequence between -75 and +1 positions, only a selection protein such as β-interferon will be produced ( 3). Or if the selection gene has two translation end sites fused to the polyhedrin promoter with a polyhedrin ATG signal at +1 and a selection gene ATG signal between +3 and the end of the polyhedrin coding sequence, the hybrid and Both select proteins will be produced. However, the proportion of protein produced may vary depending on the position of the secondary ATG signal and the characteristics of the selector sequence. Optionally, if a gene is fused to a polyhedrin promoter without its own ATG initiation site, it may be necessary to fuse either the synthetic ATG or polyhedrin ATG translational initiation signal to the protein coding sequence to maintain an accurate translation reading frame for the selected gene. have. The protein product produced depends on the factors described above.

Deposition of Transfer and Recombinant Expression Vectors

The recombinant baculovirus expression vector Ac380-IFN-β was deposited on 13 May 1983 by ATCC (Rockville, Maryland) and assigned accession number ATCC 40071. The modified polyhedrin transfer vector pAc380 plasmid in E. coli K-12 and the recombinant transfer vector pAc380-IFN-β in E. coli K-12 were published in the Agricultural Research Culture Collection (Peoria, Illinois), 1983. It was deposited on 13 May and repaired under accession numbers NRRL B-15428 and NRRL B-15427 respectively. The modified polyhedrin transition vector pAc373 was deposited with the Agricultural Research Cultures Association on May 7, 1984, and repaired under accession number NRRL B-15778. On the other hand, E. coli pAc 380 and E.coil pAc373 have been deposited on 27 August 1984 with the Accession No. KFCC-10106 and KFCC-10107 respectively.

Starting Materials and Methods Plasmids

The plasmids used in the examples below are pBR325 and pUC8 from E. coil and can be obtained from Bethesda Research Labs, Gaithersburg, Maryand.

Viral DNA

AcNMPV, strain M3 and AcMNPV strain E2 and the wild form used in the examples as the original source of viral DNA are 89 Virology 517-520) 1978 by GESmith and MDSummers and 39 J.virol by GESmith and MDSumers. It was isolated in this laboratory by the technique described in 125-137 (1981).

IFN-βDNA

The DNA fragment containing the INF-β gene used in the Example was isolated from plasmid pBR13 and this plasmid was obtained from Dr. John Collins (Gesellschaft fur Biotechnologische Forschung (GBF), Braunschweig Stock-hein, West Germany).

CAT DNA

DNA fragments containing chloramphenicol acetyltransferase (CAT) were isolated from plasmid pBR328 obtained by Drs. Bernard Moss and Mark Cochran, National (Cander Institue, Bethesda, Maryland).

IL2 DNA

DNA fragments containing the coding gene of human interleukin-2 (IL2) were isolated from plasmid PIL2-2B obtained from Drs. Grace Ju and Peter Lomedico, (Hoffmann LaRoche Research Center, Nutley, New Jersey).

Bacteria cells

E. Coli JM83 used to clone pUC8 plasmid in the examples was obtained from Bethesda Research Labs, Gaithersburg, Maryland.

E. coli RR1 used in the examples to clone the pBR325 plasmid was obtained from Dr. Savio Woo, Baylor (College of Medicine, Houston, Texas).

enzyme

The following and restriction endonucleases were obtained from Bethesda Research Laboratories, Gaithersburg, Maryland and used according to the supplier's recommendations. : EcoRI, Xhol, BamHI, SmaI, PstI, BstEII, EcoRV, KpnI and HindIII. The following enzymes were also obtained from Bethesda Research Laboratories: Calf's alkaline phosphatase (CAP) DNA ligase, Bal 31 exonuclease, S1 nuclease and T4 polynucleotide kinase.

Standard methods used according to the cloning procedure described in the Examples are described in T. Maniatis. Molecular Cloning: A Laboratory, Cold Spring Harbor Laboratory, 1982 by E.F.Fritsch and J. Sambrook. The description includes procedures of the following standard methods: cloning to E. coli plasmids, modification of E. coli cells, plasmid DNA purification, phenol extraction of DNA, ethanol precipitation of DNA, agagel electrophoresis, agagel Purification of DNA fragments from, restriction endonucleases and other DNA-modifying enzyme reactions, in all cases DNA is purified by phenol extraction and ethanol precipitation.

Virus stocks used in the examples are prepared and titrated in Spodoptera purgiferda in 10% calf serum and TNM-FH medium (see W.F.Hink, 226 Nature (London) 466-467 (1970)).

Methods for culturing viruses and cells are described in LE Volkman and MDSummers, 19 J. Virol., 820-833 (1975) and LEVolkman and M. DSummers, CHHsieh, and 19 J. Virol 820-832 (1976). . Viral growth kinetics are determined using S. pruperferda and 1.5% agar as described by Volkmen, et al., Supra.

Example 1

Construction of polyhedrin transition vector

To prepare a polyhedrin transition vector according to the present invention, a DNA fragment containing the AcMNPV polyhedrin gene is cloned into the EcoRI region of plasmid pUC8. This is done using the AcMNPV plaque purified strain, referred to as M3 (GESmith MDSummers, 89 Viology 517-527 (1978)) as the original source of viral DNA, extracting AcNMPVPNA from the virus and presenting it to Smith and Summers. Purification by equilibrium centrifugation at cesium chloride density gradients as described in the above reference. AcMNPV DNA is completely digested with EcoRI-limited enonucleases The resulting AcMNPV-EcoRI-I fragments are first introduced into pBR325 to form pI10. After cloning it is subcloned into pUC8 using standard cloning methods to form PI8 (see Figure 1).

Recombinant plasmid pI8 has three BamHI recognition sites (see Figure 1): one at position 4210 of the polyhedrin gene, one at position 7300 of pUC8 and one at position 6160 of the EcoRI-I fragment. Positions 6160 and 7300 can be removed from pI8 so that a target gene, such as a typical CAT, IL2 or IFN-β gene, can be easily cloned into pI8 at the target position (4210) adjacent to the polyhedrin promoter. The pUC8 BamHI restriction site, located at about 7300 of pI8, not located in the polyhedrin gene, is removed by the following method: 10 μg of pI8 is digested with Pst I and SmaI, the DNA is purified, and the S1 nuclease buffer (0.03 M sodium acetate, pH 4.4, 0.25 M NaCl in 0.0045 M ZnCl ₂ ) and resuspend in 500 ml of s1 nuclease 1 ml mixture. After incubating the reaction mixture for 15 minutes at 37 ° C, DNA is electrophoresed on 0.8 agarose gel. Polymer linear DNA is purified on gels and used to transform E. coli JM83 cells into ampicillin resistant (am ^r ) galactosidase negative (gal ⁻ ).

Plasmids that lost PstI, BamHI, SmaI and EcoRI sites in the pUC8 cloning region were isolated. This plasmid is called pI8SPS (see FIG. 1).

The BamHI site at position 6160 (Smith, GE, JMVlak and MDSummers, J. Virol. 45-215225) of AcMNPV EcoRI-I (part of pi8SPS) is removed by the following method: 10 μg of pI8SPS with 10 units of BamHI. Together in 50μl buffer solution and incubated at 37 ℃. After 3,6,12,20,30 minutes, take 10 sample samples and terminate the reaction by adding EDTA to 10 ^mM . Collect 10 μl aliquots and electrophores at 0.7 agarose. Full length linear DNA is isolated from the gel, treated with the above S1 nuclease and circularized with T4 DNA ligase. JM83 cells were transformed with am ^r gal ⁻ , and the plasmid to be prepared was confirmed to have lost the BamHI restriction site at position 6160. This plasmid had one BamHI cloning site at 4210 sites located at +175 bases from the translational start of the polyhedrin gene and was named pAc101, the "parent" AcMNPV polyhedrin gene transfer vector (see Figure 1).

Example 2

Transformation of the transition vector

In addition to pAc101 a large number of transition vectors have been constructed to determine suitable positions in the polyhedrin gene for insertion of a selection gene (see Figure 2). These transfer vectors delete oligonucleotide having a BamHI recognition site at the deletion site by deleting some or all of the DNA sequence encoding the 86 amino-terminal residue of the polyhedrin and the 5 'non-transcriptional polyhedrin mRNA sequence by one of the following methods. It is made by inserting a nucleotide synthetic DNA linker.

In the same manner as described in Example 1, 4210 is cloned into pUC8 with EcoRI to BamHI fragment 0 obtained from pi 10 and the resulting plasmid is named p8 '(see also FIG. 2). In addition to the polyhedrin gene, this fragment contains up to +175 polyhedrin genes and about 4000 base pairs of the AcMNPV DNA sequence. The deletions around the BamHI site at position 4210 are introduced into pB 'in the following manner (see Figure 2): 40 μg of pB' is digested with BamHI, followed by purification of DNA (pure separation, 0.7% agarose). Electrophoresis). Extract the linear DNA fragment from the gel and incubate for 30 minutes at 30 ℃ in 100μl buffer solution with 0.5 units of Bal 31 exonuclease. Take 10 μl at intervals of 1,2,5,10,15,20,25,30,35,40 minutes to make a sample sample and terminate the reaction by adding 10 μl of 0.25 M EDTA to each sample sample. Collect each sample sample to purify DNA. The ends of the DNA are recovered by incubating for 30 minutes at 23 ° C. in 100 μl of buffer with 4 units of E. coli DNA polymerase (Clenob fragment). DNA was purified and 1 μg of phosphorylated BamHI linker (5′-pCGGATCCG-3 ′) was mixed with 20 units of T4 DNA ligase in 100 μl of the reaction mixture. After incubation at room temperature for 2 hours, DNA is purified. Next, the DNA particles are resuspended in a mixture of 100 μl of buffer and 20 units of BamHI and digested at 37 ° C. for 4 hours. The digested DNA is electrophoresed at 0.7% agarose and the pB ′ cleaved (incomplete) plasmid with deletions up to 800 base pairs is purified from the gel. DNA was circularized with T4 DNA ligase and used to transform JM83 cells with am ^r , gal ⁻ . The resulting clones constitute a "library" of variant plasmids named pB'Bal 1,2,3, etc. based on the location of the BamHI recognition site. Several pB'Bal deletion plasmids are selected and the fragments from each of XhoI (1900) to BamHI linker (at positions 4000 to 4210) are purified from Aza Rosegel (fragment A) (Figure 2). ). Fragments from XhoI (1900) to BamHI (4210) were removed in pAc101, and the remaining sequences were purely separated from the colloid (B powder) (Figure 2). 0.1 μg of each A powder is mixed with 0.1 μg of B powder, incubated in 20 μl of buffer solution with T4 DNA ligase 1 unit, and then used to transform JM83 cells with am ^r , gal ⁻ . The resulting plasmids were modified transition vectors, for example, those derived from pB'Bal ₁₁ were named pAc311, those derived from pB'Bal60, pAc360, and the like. In this way, a set of "modified" transition vectors were constructed representatively with the BamHI cloning site at positions between +175 and -100 of the polyhedrin gene. In FIG. 3, the BamHI recognition site positions in the parental transition vector pAc101 are shown the BamHI recognition site positions in the four modified transition vectors, pAc380, pAc373, pAc311, and pAc360.

Example 3

Construction of a Manufacturing Transfer Vector Containing Polyhedrin-IFN-β Gene

Any transition vector prepared according to the method of Example 1 or 2 can be used for the construction of a recombinant transition vector in which the gene of interest is linked to a polyhedrin gene at various positions, depending on the particular modified transition vector used. In order to construct a plasmid named pAc380-IFN-β, a process of incorporating IFN-β gene encoding human β-interferon synthesis into pAc380, a transformation vector at a single BamHI cloning site, using standard cloning techniques described above 4 is shown schematically.

IFN-β gene is named as human IFN-β (pBR13). Reference. IFN obtained from genomic clones of H.Hauser.et.al, 297 Nature (London) 650-654 (1982) and G.Gross.et.al., 9 Nucleic Acids Res.2495-2507 (see 1981), IFN It can be specified as a 0.767 kilo base HincII fragment containing the entire protein coding sequence for -β, three additional bases prior to the ATG translational start signal and the untranslated 3 'sequence parent included for polyazenylation. Nucleotide sequences for IFN- and positions of various transcription translation signals are described by R. Dorynck, et al., 285 Nature (London) 542-547 (1980): G. Gross, et al. 9 Nucl. Acids., Res 2495-2507 (1981). ); And Sohno & T. Taniguchi, 78 Proc. Natl Acad. Sci, USA 3505-3509 (1981). The HincII DNA fragment containing the IFN-β gene is inserted into a useful BamHI restriction site of pAc380 using a synthetic oligonucleotide linker on the IFN-β gene fragment, which is in close proximity to the AcMNPV polyhedrin promoter and is a polyhedrin As in the gene, it's from 5 'to 3'. In this same manner, IFN-β genes were expressed as pAc101 and pAc101, respectively, to produce recombinant transfer vectors named pAc311-IFN-β, pAc360-IFN-β, pAc373-IFN-β, pAc101-IFN-β, respectively. It was cloned into the remaining modified transfer vectors, pAc311, pAc360 and pAc373. Potentially this gene or any other selection gene can be cloned into a transition vector having a BamHI recognition site, or other appropriate restriction endonuclease cleavage site inserted at any position within the transition vector. Appropriate inhibitory endonuclease cloning sites can be introduced at any position in the AcMNPV genome that can be fused into the plasmid as outlined in Example 1, thereby removing part or all of the polyhedrin structure sequence for insertion of the BamHI recognition site. There is no need to delete it.

Example 4

Transfer of Polyhedrin-IFN-β Gene to AcMNPV Genome

Any of the recombinant transfer vectors prepared by the method of Example 3 can be used to transfer the IFN-β gene into the AcMNPV genome to prepare a recombinant baculovirus expression vector. Metastasis is carried out by transfection in the presence of Ca ⁺⁺ and susceptible insect cells such as S. pruperferda. The method described in 52 Virology 456-467 (1973) by FLGraham and AJVan Der Eb is modified as follows: Recombinant transfer vector DNA, in particular 1-10 μg of the recombinant transfer vector pAc380-IFN-βDNA, is extracted from AcMNPV. 1-HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffered saline (pH7) containing 15 µg of carrier calf thymus DNA per ml .05) up to 950ul. The mixture forms a precipitate for 30 minutes of Vortex. 1 ml of precipitated DNA is in this case added to S. pruperferda, a susceptible insect host cell in 2 ml medium in a 60 mm culture dish. After 4 hours, the single cell layer with medium is washed off and incubated in 2 ml of 10% calf serum medium for 3 days. The resulting progeny virus forms a mixture of recombinant and nonrecombinant AcMNPV viruses.

Example 5

Selection of Recombinant AcMNPV Expression Vectors

It is necessary to separate the AcMNPV recombination expression vector from the mixture of the non-recombinant and recombinant baculoviruses obtained as described in Example 4. Isolation of these recombinants, which eliminated all or part of the polyhedrin structural genes, is characterized by (1) no polyhedrin produced by these viruses and (2) a non-combined viral form lacking a polyhedrin coat. Exploits the fact that they are viable and infectious in insect cell cultures, and recombinant recombinant vectors pAc101-IFN-β pAc311-IFN-β, pAc360-IFN-β for recombinant AcMNPV viruses in which all or part of the polyhedrin structural gene has been deleted. Recombinant AcMNPV virus obtained by transfection with pAc373-IFN-β pAc380-IFN-β was isolated by the following method: Cells present in the medium after 3 days of track specification were analyzed by LEVolkman, KDSummers & CHHseieh. Used in the standard AcMNPV polyhedrin plaque assay described in J. Virol 820-832 (1976) The developed plaques were cells infected with non-recombinant AcMNPV causing viral occlusion. Or from cells infected with recombinant aCMNPV virus that does not cause virus occlusion.The latter form of plaques (0 ^- plaques) displays and examines the former (0 ⁺ plaques) in their properties observed with a low-learning twin microscope, in this case can be observed in the nucleus of infected if the individual biruseu occlusion cell 0 ^- plaque purified biruseu in the plaque and, recombinant AcMNPV to the vector to ensure that the reserved insertion of the heterologous gene in the proper position of the EcoRI-I The DNA was analyzed with an appropriate restriction enzyme No changes in the virus genome were detected Standard infectious insect cells were obtained using standard methods to obtain large amounts of the desired recombinant virus.

Example 6

Preparation of β-Interferon Using AcMNPV Recombinant Expression Vector

Susceptible host insect cells should be infected with recombinant baculovirus to produce the desired gene product. The following procedure is used to prepare a typical AcMNPV expression vector by recombination with the recombinant transfer vectors pAc101-IFN-β, pAc311-IFN-β, pAc360-IFN-β, pAc373-IFN-β and pAc380-IFN-β. See also). First, susceptible S. pruperferda host cells are grown in suspension culture or monolayer at a concentration of 1 to 5 × 10 ⁶ cells / ml (cell concentration for optimal expression can be varied for each different AcMNPV expression vector. have). Remove growth medium and replace with medium containing plaques ranging from 0.1 to 100 (optimum concentration varies) forming a recombinant viral unit such as pAc380-IFN-β (figure 5) per cell at 23 ° C. for about 1 hour. do. The inoculum is left in the cells or removed or replaced with an appropriate amount of fresh medium. The protein obtained from the cloned IFN-β gene was infected with an Ac-380-IFN-β expression vector from about 12 hours post infection to 3 days post infection. Produced in Prughiperda cells. The entire infection process, including viral protein synthesis, viral assembly and cell lysis, is completed in about 72 hours. There is a significant decrease in protein synthesis between 50 and 60 hours after infection, and cytolysis is first detected about 50 hours after infection. Kinetics for the expression of the IFN-β gene vary depending on where the host cell and gene are cloned into the polyhedrin gene.

Table 1 summarizes the kinetics of IFN-β expression for each recombinant virus. A significant concentration of interferon is detected in cells infected with each expression vector up to 48 hours after infection. Among the expression vectors detected, Ac373-IFN-β and Ac380-IFN-β show the highest titers of interferon activity (Table 1). Intracellular concentrations of interferon activity were also measured and shown in Table 1. Less than 5% of the total interferon activity remains in the cells infected with Ac373-IFN-β and Ac380-IFN-β at 48 hours post infection, indicating that IFN-β secretion signals are recognized and proteins are effectively released into the medium during infection. Prove that.

Medium from Ac373-IFN-β and Ac380-IFN-β infected cells had approximately 10,000 IU / ml of interferon at 12 hours post-infection and increased to nearly 5 × 10 ⁶ IU / ml by 42 hours after infection. do.

TABLE 1

The kinetics of interferon expression in S. pruky Perda cells infected with various expression vectors were investigated. The test results are as follows.

The synthesis of polyhedrin in AcMNPV infected cells is known to follow a similar transient expression. Interferon production is low in Ac360-IFN-β and interferon production is less in the cells infected with Ac311-IFN-β virus, but more than 95% of the activity is present in the medium (Table 1). Relatively low concentrations of interferon are detected in Ac101-IFN-β infected cells, most of which are intracellular (Table 1). The titers of recombinant virus infected cells range up to 3-8 × 10 ⁸ plaque forming units per milliliter of medium, typical of AcMNPV infected cells. Inserting the IFN-β gene into the polyhedrin gene does not have any significant effect on the reproduction of the virus. For this experiment, 2 × 10 ⁶ S. pruperfera cells were generated in 5 × 10 ⁶ interferons, or 5 × 10 ⁶ IU-ml infected cell media, which are international standard amounts of human interferon. Treatment with IU / ml interferon for 12 hours. Treated cells are infected with AcMNPV or Ac380-IFN-β 100 plaque forming units. Exposing the cells to interferon has no measurable effect on the number of virus plaques developed.

Viral plaque-reduction assays in human amnion WISH cells invaded with Vesicular Stomatitis virus are used for the analysis of interferon activity. If the virus particles were removed by centrifugation, no interferon activity was measured from AcMNPV infected cells in the medium. However, if a medium containing AcMNPV virus is used during the interferon assay, interferon 1000 to 3000 reference units (IU) / ml are produced, indicating that AcMNPV virus particles clearly induce endogenous interferon in WISH cells. will be.

These results were predicted because many species of enveloped viruses are known to induce interferon production in human cells. To eliminate this effect, all the samples below are centrifuged before the experiment.

Serum albumin (6 mg / ml) in medium (WfHink, 226 Nature) (London) 446-467 (1970) for expression of IFN-β in S. pruperferda cells infected with Ac380-IFN-β. Experiments were conducted to determine whether or not calf serum (10%) was required. Eight hours after infection, the medium was replaced with Grace's medium containing 0-10% calf serum (without serum albumin, see TCCGrace, 195 Nature (London) 788-789 (1962)). Let's do it. Interferon activity is determined for each modified medium 48 hours after infection. Without serum there was a 10-fold decrease in interferon activity. Addition of 0.5% serum resulted in the same concentration of interferon activity as the control containing 10% serum.

The specific activity of IFN-β in Ac380-IFN-β infected cells was about 5x10 ⁶ IU per protein when produced in medium of Grace containing 0.5% serum. Assuming that the activity of purified β-interferon is 2 × 10 ⁸ IU / ml (E.knight Jr., 73 Proc. Natl. Acad. Sci. USA 520-523 (1976)), β-interferon is in the medium. Will account for about 1% of the total protein. Reanalysis of the measured interferon activity in the medium of Ac373-IFN-β and Ac380-IFN-β infected cells reveals two polypeptides with molecular weights of 17,000 (17K) and 20.5K.

It has been reported that the size of aglycosylated and glycosylated human IFN-β proteins may correspond to 17K and 20.5K polypeptides, respectively. At 30 hours post-infection, 17K polypeptides were being generated in Ac360-IFN-β infected cells, and at 48 hours post infection both 17K and 20.5K polypeptides were detected. Only 17K polypeptide could be detected in Ac311-IFN-β infected cells 30 and 48 hours post infection. Sufficiently produced 23.5K protein was observed in Ac360-IFN-β infected cells.

This size contains 21 amino acid coding peptides and 14 amino acids derived from the first 10 codons of the BamHI linker (connector) sequence of the polyhedrin gene and is expected to be the size of the hybrid protein that constitutes the total interferon protein (-13-). do.

17k and 20.5k proteins formed in Ac380-IFN-β and smaller levels of Ac380-IFN-β-infected cells reacted with antibodies from human IFN-monoclonal clones (IFN-β-monoclonal antibodies were identified by PWTrown and Hoffmann). -Provided by La Roche Inc.). This antibody tends to significantly reduce the reaction with 20.5k protein compared with the 17k protein. This was partly due to 17k accumulating at higher levels intracellularly than 20.5k. The antibody may also respond to epitopes of 17k polypeptides that may be partially obscured, such as by glycosylation of 20.5k polypeptides. The alleged hybrid 23.5k and 32k proteins also reacted with IFN-β-antibodies. Polyclonal antibodies against polyhedrin detected 57 amino acids of 32k protein and 10 amino acids of 23.5k protein expected from DNA sequence at the N-terminal end of the hybrid protein. Ac380-IFN-β-infected cells were labeled [3H] mannose at the end of infection to demonstrate that 20.5k IFN-β was glycosylated.

The major mannose-containing glycoproteins labeled in Ac380-IFN-β-infected cells were 20.5k IFN-β and three other proteins.

Example 7

Construction of Chloramphenicol Acetyl Transporase Gene and AcMNPV Recombination Transition Vector

The E. coli potential element Tn9 contains the gene of chloramphenicol acetyltransferase element (CAT), which always confers resistance to chloram phenylcol. Expression in eukaryotic vectors is known as an easy way to measure the expression of promoters in animal cells (see Mackett, M., G. Smith and B. Moss 1984. J.virol 49: 857-864 and reference books). .

AcMNPV-CAT expression vectors are useful in experiments designed to optimize the production of heterogenes in baculovirus vectors.

A 770 base pair TaqI DNA fragment containing the CAT-coding sequence is isolated from pBR328 and cloned into the AccI site of pUC7. The CAT-coding sequence is cleaved with BamHI and inserted into the BamHI site in pAc373. The obtained plasmid transition vector is designated pAc373-CAT.

Example 8

Construction of Ichin-2-gene and AcMNPV Recombinant Transfer Vector as Human

Human interleukin-2 (IL2) is produced in small amounts in human lymphocytes stimulated by mitotic substances and antigens. IL-2 was originally described as a factor that enables long-term growth of T lymphocyte cells in culture (Morgan, DA, Ruscetti, FW and Gallo, R., Science 193: 1007-1008 (1976). It has been suggested to play a pivotal role in the stimulation and maintenance of responses and to be involved in certain human diseases (Altman A., Theofilopoulos, AN, Weiner, R., Katz, DH and Dixon, FJ, J. Exp. Med. 154: 1403-1417 (1981).

The use of AcMNPV expression vectors for mass production of IL-2 greatly facilitates clinical diagnosis and therapeutic manipulation of the human immune system. Recently, several laboratories have reported the production of biologically active IL-2 in bacterial cells using gene isolation and plasmid vectors of IL2 (Roseberg, et al. Science 233: 1412-1415 (1984) and its See note).

A 1000 base pair of BamHI (fragment) comprising the IL2 coding sequence is isolated from PIL2-2B and inserted into the BamHI site at pAc373 and pAc380. The plasmid transfer vectors produced are referred to as pAc373-IL2 and pAc380-IL2.

Example 9

Transfer of Polyhedrin-IL2 and CAT Genes to AcMNPV Genomics

Transfer of the polyhedrin-IL2 and CAT genes to the AcMNPV genome and selection of the recombinant AcMNPV expression vector is performed as described in Example 4. AcMNPV expression vectors prepared from pAc373-IL2, pAc380-IL2 and pAc373-CAT are referred to as Ac373-IL2, Ac380-IL2 and Ac373-CAT, respectively.

Example 10

How to produce IL2 using AcMNPV expression vector

S. pruperfera cells are infected with Ac373-IL2 or Ac380-IL2 expression vectors as described for Ac380-IFN-β. After 48 hours of infection, the medium and infected cells were harvested and the biological activity level of interleukin was measured by Gillis, et al. J.Immunol. 120: measured using the assay described by 2027-2032 (1978).

Using this assay the specific activity of IL2 was measured in 1 × 10 ⁸ units per milligram of protein.

Both expression vectors produce high levels of interleukin activity, but Ac373-IL2 produces about four times more interroingin than Ac380-IL2 (see Table 2).

About 80% of interleukin activity is present in the medium, demonstrating that the protein is effectively isolated from cells in infectious diseases. In the isolation experiment performed by Dr. Grace JU. Hoffmann-La Roche research center, S.purgiperda cells were infected with Ac373-IL2 and Ac380-IL2, and interleukin activity levels were 24, 48 hours after infection. Measured in the medium after 72 hours. Fresh medium is applied at 24 and 48 hours.

In fact, all activity is produced between 0 and 24 hours and 24 to 48 hours after infection (see Table 2).

Judging from the specific activity of IL-2, it is estimated that at least 1 mg of protein is produced, which is secreted per liter of AcMNPV infected cells.

Analysis of proteins synthesized in Ac373-IL2 and Ac380-IL2 infected cells is performed.

Two proteins abundantly produced by the AcMNPV expression vector are not produced in AcMNPV infected cells.

The new protein is about 15.5k and 16k Daltons in size. This is consistent with the size of the interleukin predicted from the DNA sequence of about 15.5K (assuming 20 amino acid signal peptides were removed at the amino-terminus of the protein).

Table 2 shows the production of interleukin-2-activity in S. pruperfera cells infected with AcMNPV expression vectors. Samples of the medium and infected cells at infection number 1 were collected 48 hours after infection. Media samples at infection number 2 were collected 24 hours, 48 hours and 72 hours after infection and fresh media were applied 24 and 48 hours after infection.

Infection number 1 ^a

Infection number 2 ^b

Manufactured in ^a Texas ^A & University.

^b Manufactured by Hoffmann-La Roche Ressearch Center.

Example 11

Production of CAT Using AcMNPV Expression Victor

S. pruperfera cells were infected with Ac373-CAT as described in Ac380-IFN-β and CAT enzyme activity was measured in cells and medium as described by Mackett, et al. IBID. 24 hours after infection. . ACT enzymatic activity could not be detected in uninfected cells or cells infected with AcMNPV. However, high levels of activity are produced by Ac373-CAT in both the cells and the medium.

Analysis of the protein synthesized in Ac373-CAT infected cells is carried out. A new protein of 27k Daltons, which is not made in AcMNPV infected cells, is produced by this expression vector. The size of CAT is predicted to be about 27k from the DNA sequence. Abundant 27k protein is present in both infected cells and medium. From the amount of protein observed on polyacrylamide gels it was estimated that about 40 mg of CAT enzyme was produced per liter of Ac373-CAT infected cells.

The invention described above and its advantages and possibilities have been recognized, and only some preferred examples of the invention have been described. Many variations in the materials, methods and amounts used may be made without departing from the scope and subject matter of the invention or the advantages of the invention. It will also be appreciated that the present invention has the advantageous use that any kind of selection gene can be used for insertion at any of several positions in the baculovirus genome.

For example, isolation of recombinant AcMNPV viruses in which all or part of the polyhedrin gene has not been deleted allows the present invention to be used in a variety of ways. The use is that the occlusal polyhedrin coating of AcMNPV virus is very resistant to external influences.

For example, as described in Example III, the selection gene can be cloned into the virus genome at a location other than the polyhedrin gene. In particular, it can be cloned in a position that can be regulated by the 10k promoter so that the expression of the selector is expressed at a higher level. Recombinant AcMNPV viruses can be isolated and used as stable expression vectors.

The stable expression vector can be used as a stable form capable of transferring the recombinant AcMNPV virus with appropriate host cells and sufficient medium.

Expression vectors can be used in pest control systems by selecting genes that produce proteins that are toxic to certain host insects or a wide range of host insects and cloning the genes into AcMNPV expression vectors (the possibility is LKMiller, et al., 219). Science 715-721 (1983).

Recombinant expression vectors can be applied to plants or animals when insects are pests. As described above, when the insects are ingested by insect pests, the occlusal bodies are separated from the intestinal lumen, and the recombinant viruses invade barrier cells and start replication.

During replication, when a gene of a protein that is toxic to pest insects is expressed and the insect ingests a wild form of AcMNPV virus, the insect becomes incapable or dies within a short period of time. Eventually it dissolves. Experiments in this and other laboratories indicate that the expression of 10k protein is induced rapidly to 24 days after infection and to high levels at about 48 hours. As a result, the coding genes of the necessary insect toxin cloned into the AcMNPV genome under the control of the 10k promoter will be expressed over time. Insect killing or incapacity can be expected soon after the expression of the gene of choice, and the degree of damage to plants and animals infested by pests is reduced when compared to the infection of insects with wild-type baculoviruses.

The genes can be fused into polyhedrin sequences and inserted into the baculovirus genome when the polyhedrin coating is degraded by the intrinsic alkaline state of the insect, and then the toxic gene product is released.

By using the present invention, the recombinant gene is not expressed in insect enterocytes and the insect is detoxified.

In addition, higher levels of gene expression than the levels of gene expression measured in the above examples may be possible using the present invention. For example, the INF-β gene (or other gene) can be cloned into the baculovirus genome more than once. In particular, copies may be inserted such that the expression is under the control of the polyhedrin promoter and other copies may be inserted such that the expression is under the control of the 10k promoter and then several copies may be inserted at many different restriction sites and each copy may be And other DNA sequences recognized as promoters by a promoter of or baculovirus.

When infected into susceptible insect cells, the amount of interferon (or other polypeptide) produced can increase significantly above the measured level.

Modifications of the invention described herein may be made by those skilled in the art, and such modifications also fall within the scope of the invention as defined by the appended claims.

Claims (22)

The baculovirus DNA is cut to prepare a DNA fragment containing the baculovirus gene or portion thereof, the DNA fragment is inserted into a cloning medium, and then at least one of the selection genes or portions thereof is inserted into the modified cloning medium. The selection gene or a portion thereof produces a recombinant transfer vector under the transcriptional control of the promoter of the baculovirus gene or its own promoter, and the recombinant transfer vector is contacted with the baculovirus DNA to induce recombination. A non-recombinant baculovirus mixture is prepared, and a recombinant baculovirus expression vector is separated from the mixture, thereby producing a recombinant baculovirus expression vector capable of expressing a selection gene or part thereof in host insect cells. How to. The method of claim 1, wherein the selection gene or portion thereof is inserted into the cloning medium in place of at least a portion of the baculovirus gene. The method of claim 1, wherein the baculovirus gene is a polyhedrin gene comprising a polyhedrin promoter or a portion thereof. The method of claim 3, wherein the selection gene or portion thereof is inserted into the cloning medium in place of at least a portion of the DNA sequence encoding polyhedrin synthesis. The method of claim 1, wherein the baculovirus is a 10k gene comprising a 10k promoter or a portion thereof. The preparation according to claim 1, wherein the baculovirus is Auto grapha californica, Trichoplusia ni, rachiplusia ou or Galleria mellonella. Way. The method of claim 1, wherein the baculovirus is Autographa californica. The baculovirus DNA is cut to produce a DNA fragment comprising the baculovirus gene or a portion thereof, the DNA fragment is inserted into a cloning medium to prepare a baculovirus gene transfer vector, and at least one selection gene or At least one selection gene inserted into the baculovirus genome, wherein the portion is inserted into the baculovirus gene transfer vector so that the gene or portion thereof is subjected to transcriptional control of the baculovirus promoter or its own promoter or Method for producing a recombinant transfer vector having the portion. The method of claim 8, wherein the selection gene or portion thereof is inserted into the cloning medium in place of at least a portion of the baculovirus. The method of claim 8, wherein the baculovirus gene is a polyhedrin gene comprising a polyhedrin promoter or a portion thereof. The method of claim 10, wherein the selector or portion thereof is inserted into the cloning medium in place of at least a portion of the DNA sequence encoding polyhedrin synthesis. The method of claim 8, wherein the bacteriovirus gene is a 10k gene comprising a 10k promoter or a portion thereof. The method of claim 8, wherein the baculovirus is Autographa californica, Trichoplusia ni, Rachiplusia ou, or Galleria mellonella. . The method of claim 8, wherein the baculovirus is Autographa californica. The DNA fragment may be cut to produce a DNA fragment comprising at least one promoto, and to prepare a modified cloning medium having at least one useful site capable of cloning the baculovirus DNA and cloning the selection gene or part thereof. Consisting of insertion into a cloning medium, wherein said useful cloning site has a selection gene or portion thereof inserted, which can be used as an intermediate medium for baculovirus gene manipulation, characterized in that it is under transcriptional control of the promoter. Method for producing a viral transfer vector. The method of claim 15, wherein the baculovirus gene is a polyhedrin gene or portion thereof containing a polyhedrin promoter. The method of claim 15, wherein the bacterium is a 10k gene or portion thereof comprising a baculovirus gene 10k promoter. 16. The method according to claim 15, wherein the baculovirus is autographer Carylponica, Tricoprucciani, Laprussia ow or Galleria melonelline. 16. The method of claim 15, wherein the baculovirus is autographer california. In a method of synthesizing a selection polypeptide by infecting a sensitive host insect cell with a recombinant baculovirus expression vector, the expression vector is a recombinant baculovirus genome comprising at least one selection gene or part thereof, and the selection gene or The part is present under the transcriptional control of the baculovirus promoter or its own promoter. The method of claim 20, wherein the baculovirus promoter is a polyhedrin promoter. The method of claim 20, wherein the baculovirus motor is a 10k promoter.

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