JPH0573393B2 - - Google Patents

Description

[Detailed description of the invention]

(Objective of the Invention) An object of the present invention is to provide a method for producing substances useful for medicines, etc. by genetic engineering techniques, and in particular to provide a method for producing substances more efficiently than conventional methods. There is also a virus
The purpose is to provide a method for producing substances in vitro or in living organisms using DNA, and a method for efficiently producing various useful substances using nuclear polyhedrosis virus in silkworms. Our goal is to provide the following. Moreover, the present invention
Vectors and recombinant viruses useful in these methods
Our objective is to provide DNA and methods for their production. Another object of the present invention is to provide vectors, recombinant viral DNAs, and methods for producing them that are useful in these methods. (Prior Art) Many methods have been reported for producing useful substances using Escherichia coli, Bacillus subtilis, yeast, etc. using plasmids and the like through gene recombination technology. Also, viral DNA
(Autographa californica nuclear
polyhedrosis virus DNA) to replace its structural genes in cultured cells (Spodoptera
An attempt to produce β-interferon and β-galactosidase in established cultured cells of M. frugiperda was also reported (Molecular and
Cellular Biology 3 (12)2156-2165 (1983), 4 (3)
399–406 (1984). However, this method has a problem in that it uses A. californica, which is a pest that lives outdoors, and is not satisfactory as a method for producing useful substances. The present inventor completed the present invention as a result of research in order to provide an even better method for producing useful substances. (Structure of the Invention) The present invention relates to Bombyx Nuclear Polyhedrosis Virus (hereinafter referred to as Bombyx Nuclear Polyhedrosis Virus).
Regarding the method of genetically engineering the production of useful substances such as proteins or glycoproteins using the DNA of BmNPV (abbreviated as BmNPV); This invention relates to recombinant DNA obtained by recombining DNA with genes for producing useful substances, and to recombinant vectors useful for the recombination.
Furthermore, the present invention has a 5' upstream DNA fragment containing the promoter region of the polyhedral protein structural gene of BmNPV DNA, a translation initiation codon, and a gene for producing a useful substance, and further comprises a 3' downstream DNA fragment of the polyhedral protein structural gene of BmNPV DNA. This article relates to a recombinant vector for transfer that contains a DNA fragment but does not contain anything else, and a method for infecting and introducing BmNPV DNA. The present invention also provides recombinant vectors for transfer.
A mixture of BmNPV DNA can be inoculated into cultured cells or silkworm organisms to produce recombinant BmNPV, or BmNPV DNA and E. coli plasmids can be inoculated into cultured cells or silkworm organisms.
Create recombinant DNA with pBR322, and then recombine this polyhedral protein structural gene part with a gene for producing useful substances to create recombinant BmNPV DNA, and inoculate this into cultured cells or silkworm organisms in the same way. The present invention relates to a method for producing useful substances by multiplying them. Next, embodiments of the present invention will be described in more detail. BmNPV is baculovirus
Bombyx mori is a type of insect virus that is highly host specific, infecting silkworms (Bombyx mori) and accumulating large amounts of polyhedral proteins in their cells. This virus has a circular double-stranded DNA genome of approximately 140 kbp, which is extracted from the virus particle by protease treatment.
It can be obtained by conventional methods such as sodium lauryl sulfate (SDS) treatment and extraction. This circular double-stranded DNA (hereinafter referred to as viral DNA or BmNPV)
(abbreviated as DNA) is a fairly large circular DNA, so small DNA fragments are produced by restriction enzyme treatment.
Polyhedral protein structural gene and its surroundings (5′ upstream and
It is appropriate to select and use a fragment containing the 3′ downstream portion). Various restriction enzymes are known and commercially available, so any suitable one may be selected and used, and the conditions for use may be set appropriately depending on the enzyme. In addition, DNA in the technical content of the present invention
Fragment synthesis (chemical synthesis, cutting by restriction enzyme treatment), separation and search, DNA sequence analysis, processing of E. coli (transformation, culture, plasmid acquisition, etc.) uses well-known genetic manipulation techniques. You can do it by doing this. To select DNA fragments containing structural gene parts from many DNA fragments obtained from BmNPV DNA,
This may be carried out by Southern hybridization or the like using a probe prepared by a conventional method. The fragment to be selected needs to contain the structural gene part, but also have a considerable length of DNA strand on the 5' side (upstream) and in some cases on the 3' side (downstream). It is important that The length of this length is important for the production efficiency of the target useful substance, and is also related to the ease of handling described below, but it can be confirmed by experiment. Although it may be time-consuming, it will not be difficult for those with ordinary knowledge in this field to refer to the following explanation, especially the examples.
One suitable fragment for this purpose is a fragment of approximately 10.6 kbp obtained by digestion with EcoR, but various fragments obtained using other restriction enzymes may be used. Structural gene of polyhedral protein and DNA before and after it
Remove the structural gene part from the fragment containing the chain, and create an appropriate length containing the upstream promoter region.
Various techniques are used to extract DNA strands and to extract and utilize DNA strands of appropriate length downstream of structural genes. That is, the structural gene portion is identified by searching the DNA sequences of fragments obtained by treating with various restriction enzymes,
That portion can be removed by cutting with a restriction enzyme (eg, digestion with Bal31). The required upstream and downstream DNA strands may be prepared as remaining parts of Bal31 digestion, or
It is also possible to produce it by elucidating the DNA sequence and then cutting it with a control enzyme, or by chemical synthesis. Regarding silkworm polyhedral protein, S. Celebriani et al. conducted amino acid analysis and published the 244 amino acid sequence (J. Invertebrate Pathology 30
442-443 (1977)). Therefore, BmNPV-derived
By elucidating the base sequence of a DNA fragment, matching its codons and amino acids to create an amino acid sequence, and comparing this with the sequence of Celebriani et al., we can determine which part of the polyhedral gene it corresponds to, or if it is a different part. It is possible to judge whether In order to remove the structural gene portion and utilize the upstream and downstream DNA fragment portions, the present inventor has determined that the EcoR cleavage fragment of BmNPV DNA (approximately
10.6kbp) is preferred and Hind
It was confirmed that two convenient fragments could be obtained when cutting with . However, in order to achieve the purpose of the present invention, it is not necessarily limited to this fragment, and it is possible to create other suitable fragments by searching using various regulatory enzymes. it is conceivable that. In the following, examples of the above-mentioned suitable fragments will be explained as necessary. In addition, when displaying DNA sequences, etc., unless otherwise specified, the 5' side is placed on the left and the 3' side is placed on the right according to the general method, and the fragments are represented by following the control enzyme name with a hyphen (-). In this case, left and right have the same meaning. A summary of the preferred EcoR-EcoR fragments mentioned above is provided below.

[Table] If this fragment is divided into smaller fragments and searched using methods such as Southern hybridization, the polyhedral protein gene portion can be found. In this example, the presence of this structural gene was found in the Hpa I-Hind fragment (approximately 1.4 kb). Therefore, in order to utilize the upstream and downstream parts of the structural gene,
Hind-Hind (approximately 3.9kb) by Hind processing
It is convenient to prepare and use two types of fragments: Hind-Hind and Hind-Hind (approximately 3.1 kb). These two types can be separated by conventional methods such as agarose electrophoresis. It is convenient to manipulate the two separated fragments by incorporating them into separate plasmids containing artificial linkers; for example, commercially available plasmids pUC9 and
pUC8 (both Pharmacia P-L
Biochemicals) is suitable for this purpose. The downstream Hind-Hind fragment (approximately 3.1kb) is
It is used by integrating into the Hind site of pUC8. After integrating the upstream Hind-Hind fragment containing the polyhedral protein gene into the Hind site of pUC9, it is cut at one site with a restriction enzyme (e.g. UcoRI), and when the resulting DNA fragment is treated with Bal31, the DNA strand is Since one base is digested on each side of the break point, the length of the DNA chain is convenient for adjusting. By changing the reaction time to create fragments of several lengths and analyzing their base sequences, it is possible to find fragments in which the polyhedral gene has been removed. In this way, the polyhedral gene is removed and its upstream virus-derived promoter region is included.
DNA fragments can be obtained. This is combined with the downstream DNA fragment of the other polyhedral gene and incorporated into a plasmid to create a vector plasmid, and the gene for producing useful substances is inserted into the site where the polyhedral gene existed between the two fragments. It is useful for integrating to produce recombinant plasmids. It is. Here, the useful substance in the present invention means a peptide, protein, or glycoprotein. For example, the upstream part is inserted into pUC9 after Hind treatment to create a plasmid, and the downstream part is Hind-treated.
The Hind fragment (approximately 3.1 kb) is inserted into the Hind site of pUC8 to create a plasmid, and the upstream and downstream parts are integrated into the same plasmid using the common restriction enzyme site of both plasmids. In this case, designing so that an artificial linker portion exists between the two may be convenient for integrating genes for producing useful substances. In addition, if the vicinity of the translation start code and the end code of the polyhedral gene are deleted, the synthetic
It may be repaired by methods such as replenishing with DNA. These are performed using common recombinant DNA techniques. A variety of genes have been reported for producing useful substances, and the isolation and synthesis of many natural genes will likely be reported in the future, but it is easy to understand that they can be used. Probably.
For example, as useful substances, interferons (α-interferon, β-interferon,
γ-interferon), insulin, human growth hormone, somatomedins, interleukins, various lymphokines, and other peptides, proteins,
Examples include glycoproteins. Usually, a translation start codon (ATG) is added to the gene for producing this useful substance.
It is appropriate to combine them and perform the subsequent operations. Genes for producing useful substances are produced into recombinant plasmids using commonly used methods, such as by ligating linkers to both ends and incorporating them into a vector. As a result, the obtained recombinant plasmid has a virus-derived fragment, a DNA fragment containing the promoter region, and a DNA fragment downstream of the termination codon integrated upstream and downstream of the useful substance producing gene, respectively.
In the above operations, the base sequence may be reversed due to the incorporation or cleavage of DNA fragments, as well as the selection of fragments, but it is necessary to proceed by confirming that the orientation is correct at each step. It is natural that there is. Although the recombinant plasmid obtained as described above can itself be transformed into E. coli and grown, a remarkable effect can be obtained by utilizing the growth power of BmNPV. To achieve this effect, a DNA fragment containing a virus-derived promoter region and a DNA fragment below the termination codon that are present in the recombinant plasmid are extremely useful. The above promoter region consists of the translation start codon ATG of the polyhedroprotein structural gene of the Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA and the Hid approximately 4.5 Kbp upstream thereof.
It exists between sites, and this region plays an important role in promoting the translation of structural gene parts into proteins. The 5' upstream fragment containing the promoter region does not necessarily have to have the same nucleotide sequence as it exists in the original BmNPV DNA, and the same function can be expected even if there is some modification. For example, the translation start codon
Excellent effects can also be obtained by using fragments with deletions of -9 to -1 upstream of 5' or -19 to -1,
It was found that a sufficient effect could be obtained even with the deletion fragment of -29 to -1. However, those lacking up to about -80 cannot be said to be preferable. Furthermore, the effect of slightly modified base sequences can be easily confirmed through experiments, and it is also an aspect of the present invention to synthesize and utilize such fragments. There are various methods for obtaining recombinant BmNPV DNA in which a gene for producing a useful substance has been recombined using the above-mentioned recombinant plasmid. For example, one is in
This is an in vitro method, and one method uses silkworms. Alternatively, without using such a recombinant plasmid, there is also a method of creating recombinant virus DNA by creating a recombinant of BmNPV DNA and pBR322, etc., and recombining the polyhedral gene with a gene for producing a useful substance. This is one of the aspects. When cultured cells such as established silkworm cells (Bm cells (ATCC No. CRL-8851)) are infected in vitro with a mixture of BmNPV DNA and a recombinant plasmid containing genes for producing useful substances, the desired genes (useful Viral DNA (genes for material production)
Recombination to the recombinant viral DNA (recombinant
BmNPV DNA) is generated. This recombination is
This may take the form of replacing the target gene with the polyhedral protein structural gene region, or may take the form of additionally integrating one or more genes for producing useful substances into other regions of the viral DNA. In this way, if mixed infection is carried out in vitro, the recombinant viral DNA will proliferate and useful substances will accumulate.
Although non-recombinant and recombinant viruses may be present in the culture medium, it is possible to isolate recombinant viruses by common methods such as the dilution method or the plaque method. Moreover, the above-mentioned mixed infection can also be carried out using silkworms. Viruses propagated in Bm cells (a mixture of recombinant and non-recombinant viruses, or recombinants isolated therefrom) are infected in vitro, or transdermally or intracorporeally in silkworms. The desired useful substances can be efficiently produced by injecting them into the body, and these can be isolated and purified by known methods. (Effect of the invention) According to the present invention, useful peptides, proteins,
It is possible to safely and economically produce large amounts of glycoproteins. Since the peptides and proteins produced according to the present invention are produced in eukaryotic cells, when eukaryotic genes are used, modifications similar to those in the body, i.e., cleavage of the signal peptide,
It can undergo the addition of sugar chains, etc., and is expected to be far more useful than conventional genetically engineered products using bacteria. Furthermore, since these products are secreted outside the cells, their purification is extremely easy. Furthermore, due to humankind's many years of breeding experience and accumulated research, it is easy to raise silkworms in large quantities, and it is now possible to raise them using artificial feed. Therefore, it is estimated that it would be much more industrially and economically viable to produce useful products using viral vectors at the biological level rather than at the cellular level. The following is an example of producing α-IFN, a protein useful as a pharmaceutical, using the α-interferon (α-IFN) gene as a useful gene, and the mode of implementation thereof is shown, but the scope of the present invention is not limited thereto. Of course. Example 1 A Cloning and propagation of BmNPV using the plaque method Third instar silkworms were orally infected with BmNPV.
After several days, body fluids were collected. Add 1 part of fetal bovine serum to this
TC-10 medium (J.Invertebrate
Bm cells (silkworm established cells: Appl.
Environ. Microbiol. 44 227-233 (1982)), the cells were overlaid on TC-10 medium containing 0.75% agarose and 5% fetal bovine serum, solidified, and then incubated at 27°C for several days. The plaque formed on this plate was collected using a Pasteur pipette. This plaque method procedure was repeated twice to obtain a genetically homogeneous virus. BmNPV T3 strain, which is a representative strain of this virus, was used in the following experiments. After propagating the T3 strain with Bm cells, 5th instar silkworms were infected by transdermal inoculation, and after 6 days, the infected tissue containing formed polyhedra was centrifuged with distilled water and the precipitate was added to distilled water. Suspend, triturate, differential centrifuge (3000 rpm, 30 min) and 45% and 55% (w/w)
Discontinuous density equilibrium centrifugation (20000 rpm,
30 minutes). After removing the sucrose solution from this polyhedral suspension by differential centrifugation (3000 rpm, 10 minutes), the purified polyhedra were suspended in a 0.1M sodium carbonate (PH11) - 0.05M sodium chloride solution and reacted at 25°C for 30 minutes. to dissolve the polyhedra and release virus particles. This virus suspension was subjected to density gradient equilibrium centrifugation (18000 rpm, 30 minutes) of 10-40% (W/W) sucrose solution,
The virus was purified. Sucrose was removed by dialysis or differential centrifugation (40,000 rpm, 30 minutes) to obtain purified virus particles. B Adjustment of viral DNA, identification of polyhedral genes,
Cloning B-1 Extraction of viral DNA Add SDS (sodium lauryl sulfate, 1W/W%) and protease K (manufactured by Merck & Co., Ltd., 1mg/ml) to the purified virus solution.
It was incubated at ℃ for about 2 hours. Add an equal volume of phenol solution (10mM Tris-HCl buffer (PH7.6) - saturated with 1mM EDTA) to this solution, shake gently for about 5 minutes, and then shake at 12,000 rpm.
Centrifuged for 5 minutes. The aqueous layer was taken and the same phenol treatment was repeated twice. An equal amount of chloroform was added to this DNA-containing aqueous layer, gently shaken for about 5 minutes, and then centrifuged at 12,000 rpm for 2 minutes. After taking the aqueous layer and repeating the same chloroform treatment twice, the aqueous layer was mixed with 10mM Tris-HCl buffer (PH7.6)-1mM EDTA.
Dialysis was performed for about 2 days. The obtained viral DNA (ATCC No. 40188) was used for cloning of the following genes and transfection of Bm cells. B-2 Cloning of polyhedron gene Probe creation: in silkworms in the middle of the 5th instar
BmNPV T3 strain was transdermally injected for infection, and several days later, total RNA was extracted from fat body tissue by the guanidine hydrochloride method. This is oligo
dT cellulose column with polyA
The mRNA was purified. About this mRNA,
When examined using an in vitro translation system of rabbit reticulocytes, more than 90% of the total mRNA was polyhedral mRNA. Using oligo dT primer and reverse transcriptase from this mRNA,
cDNA was synthesized. In this case it contains ³² P
cDNA was labeled using dCTP as a substrate and used as a probe for searching for polyhedral genes. Cloning of EcoR fragment containing polyhedron gene: Purified DNA of BmNPV T3 strain was cloned into EcoR
The fragments were electrophoresed on a 0.7% agarose gel, transferred to a nitrocellulose filter, and hybridized with the probe described above. about
A 10.6 kb NAD fragment specifically hybridized. This fragment was inserted into the EcoR cleavage site of pBR322. That is, viral DNA
It was digested with EcoR, treated with phenol and chloroform as described in B-1, and the aqueous layer was separated. Add to this 1/20 amount of 4M
The DNA precipitated by adding sodium chloride solution and twice the volume of cold ethanol was added to a small amount of Tris buffer (10mM Tris-HCl (PH7.6) - 1mM
MEDTA). On the other hand, pBR322
After digestion with EcoR, it was treated in the same manner as above, and further treated with BAP (bacterial alkaline phosphatase).
(manufactured by Bethesda Research Laboratories)), treated with phenol and chloroform again, and precipitated by adding ethanol.
This was dissolved in a small amount of Tris buffer. Add T4-ligase to these EcoR-digested viral DNA and pBR322 and incubate at 5°C.
Reaction was carried out for 10 hours to allow binding. This is E. coli
Inserted into K12JM83. The transformed tetracycline-resistant E. coli was labeled as above.
Approximately 10.6 kb of EcoR containing the polyhedral gene was screened by colori hybridization using cDNA as a probe.
E. coli containing a plasmid containing the fragment was obtained. This was cultured, and plasmid DNA was purified using the cesium chloride method. This plasmid is named pBmE36, and Escherichia coli K12JM83DGB-0036 containing it has been deposited with the Microbial Technology Research Institute as FERM BP-813. The restriction enzyme map of the EcoR-EcoR insert of pBmE36 is shown in the figure. When the inserted DNA portion was further searched for by Southern hybridization using the above cDNA as a probe, only the Hpa-Hind fragment (approximately 1.4 kb) was hybridized. C Removal of polyhedral structural gene part C-1 Cloning of the part containing the polyhedral gene and its downstream part The Hind-Hind fragment (approximately 3.9 kb), which is the part containing the Hpa-Hind fragment mentioned above, and the downstream part of the polyhedral gene Hind− containing
The Hind fragment (approximately 3.1 kb) was inserted into the Hind cleavage site of commercially available cloning vectors pUC9 and pUC8 to create p9H18 and p8H225, respectively. C-2 Removal of polyhedral gene part After cutting plasmid p9H18 with EcoR,
It was treated with Bal31 and both sides were scraped off from the cutting point. By varying the treatment time of Bal31, fragments of various lengths were produced. This was treated with Hind and separated by 0.7% agarose gel electrophoresis.
Extract DNA from viruses of various lengths
Got the pieces. pUC9 was treated with Sma and Hind, and the previously obtained DNA fragment (having blunt ends and Hind ends) was ligated to prepare a plasmid, which was transformed into E. coli K-12 JM83. After propagating this, take the plasmid and use the nucleotide sequence from the 3' side of the inserted virus-derived DNA fragment as a primer (15base for M13).
The polyhedral gene portion of the virus was identified by the dideoxy method using the following sequencing primers. That is, the base sequence corresponding to the amino acid sequence of the polyhedral protein described in the literature by Celebriani et al.
Found in the base sequence of the DNA fragment, the translation initiation codon ATG was also determined. Among the various plasmids obtained according to the treatment time of Bal31,
p9B241 was designated as p9B241, which lacks the 29 bp upstream of the translation initiation codon ATG of the polyhedral gene and the polyhedral protein structural gene below ATG. Similarly, 82bp, 19bp, 18bp upstream of ATG
or p9B587, p9B310, p9B276 and those lacking 7bp and ATG or less, respectively.
It was named p9B585. The base sequence of this region is shown below.

[Table] D Construction of partially deleted plasmid for polyhedral structure gene Treat plasmid p9B241 with EcoR and Aat, and separately generate p8H225 [created in C-1].
Treated with EcoR, Aat, and Sca (unnecessary pUC8-derived fragments were fragmented with Sca). Both reaction solutions were treated with phenol and chloroform, respectively, and precipitated with ethanol.
The DNA was ligated and the resulting plasmid was transformed into E. coli K12 JM83. Extract the plasmid from the transformant and extract the virus-derived DNA.
Plasmids were selected in which the upstream and downstream parts of the fragments were ligated in the correct orientation. This recombinant plasmid has an ampicillin resistance marker, as shown by the cleavage images with various restriction enzymes, and a portion of the polyhedral gene from 30 bp upstream of the start code ATG to approximately 3 kb further upstream (untranslated portion including the promoter) and the same. Approximately 3.1kb downstream from the termination code of the gene (however, approximately 3.1kb downstream from the termination code)
300bp is deleted) in the same orientation as the original virus, and can be grown in E. coli. This was named p89B241. Similarly,
p9B587, p9B310, p9B276 and p9B585 as materials p89B587, p89B310, p89B276 and
p86B585 was constructed. E Insertion of α-IFN gene (i) Human genome lambda phage Charon4A recombinant library (Lawn et al., Cell 15 1157
(1978)) and searched for the leukocyte IFN gene, and extracted DNA containing the obtained α-IFN-J gene.
After treatment with Hind and EcoR, a fragment containing the α-IFN-J gene was separated by agarose gel electrophoresis, and this was ligated with pUC9 treated with Hind and EcoR. E. coli K12JM83 was transformed with the obtained plasmid. This plasmid was taken out and treated with Mst and Pvu to isolate a DNA fragment containing the α-IFN-J gene, with Sma
A linker (manufactured by Takara Shuzo) was attached. this
Plasmid treated with Sma and obtained in Section D
A recombinant plasmid was prepared by ligating with the Sma fragment of p89B241. After confirming that the α-IFN-J gene was inserted in the correct direction, Escherichia coli K-12 JM83 was transformed with this plasmid and propagated to produce a large amount of plasmid. This plasmid was named pIFN-1-B241. This is the upstream part of the polyhedral gene (approximately −
3kb to -30th), followed by the linker residues used during plasmid p89B241 construction.
α-IFN- obtained from 13bp and chromosome
Next to the 32 bp 5' untranslated region of the J gene, the α-IFN-J gene starting with ATG binds in the correct direction, and then the downstream part of the polyhedral gene (from about 300 bp downstream from the stop codon to about 3.1 kbp) ) are combined. (ii) the above Hind containing the α-IFN-J gene;
Escherichia coli K12JM83 was transformed with the plasmid obtained by ligating the EcoR fragment into pUC9, the plasmid was removed, and Hpa
containing the α-IFN-J gene.
A DNA fragment was separated and a separately chemically synthesized oligomer (CGGGCCATC) was phosphorylated using ³² P-ATP and T ₄ -polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) and annealed with another synthetic oligomer (CCGGGATGGCC). and made Reigate. This was separated and extracted by agarose gel electrophoresis, phosphorylated again using ³² P-ATP and _T4 polynucleotide kinase, and then ligated with the Xma cut fragment of plasmid p89B241 obtained in Section D to produce a recombinant plasmid. . α−
Confirm that the IFN-J gene is inserted in the correct direction, and use this plasmid to infect E. coli.
K12JM83 was transformed and multiplied to produce a large amount of plasmid, which was used as pIFN-2-B-241
And so. This follows the upstream part of the polyhedral gene (approximately 3 kb −30th bp from upstream) and the plasmid
Linker residues used during p89B241 construction
The α-IFN-J gene starting with 13 bp and ATG is bound in the correct direction, and the downstream portion of the polyhedral gene (from about 300th bp downstream from the stop codon to about 3.1 kb downstream) is also bound. Similarly p89B587, p89B310, p89B276
and pIFN-2− using p89B585 as the material.
B587, pIFN-2-B310, pIFN-2-B276
and pIFN-2-B585 were constructed. (iii) Synthesis of linker Chemical synthesis of oligodeoxyribonucleotides: Report by H. Ito et al. [Nucleic Acids
Research, 10 1755 (1982)], the oligomer was synthesized by a solid phase method using polystyrene resin. The fully protected bifunctional dimer () (approximately 100 mg) was dissolved in t-butylamine-pyridine (1:9 v/v) solution.
converted to the corresponding 3′-phosphoric diester () and condensed with the 5′-free-nucleoside resin () using the condensing agent mesitylenesulfonyl-nitrotriazolide (MSNT, 100 mg);
The unreacted 5′-hydroxyl group was acetylated with acetic anhydride-pyridine (1:9 v/v) solution.
The condensation product is 2% trichloroacetic acid (TCA)-
The detritylated product () was obtained by detritylation treatment with methylene chloride (see reaction formula:
COCH ₂ CH ₂ CONHCH ₂ C ₆ H ₄ −R ₅ part is resin part). These series of reactions were repeated until the desired sequence was achieved. To the obtained oligomer binding resin (approximately 20 mg) was added 0.5 ml of 0.5 M tetramethylguanidine-pyridine-2-aldoxime (in pyridine-water (15:4)).
The mixture was reacted at 37°C for 8 hours, and then treated with concentrated aqueous ammonia at 55°C for 8 hours. solution
Gel filtration was performed using a Sephadex G-50 fine (2.5 x 60 cm) column to obtain a DMTr oligomer.
This was further purified using high performance liquid chromatography (HPLC). The column is a reverse phase column (SSC-ODS-272, 0.6 x 20
cm) and solution A (0.02M-TEAA, PH
7.0) and solution B (50% in 0.02M TEAA, PH7.0)
DMTr oligomers were purified using a linear concentration gradient method with (acetonitrile). to this
80% acetic acid was added, treated at room temperature for 20 minutes to perform detritylation, and then subjected to the above-mentioned HPLC to obtain an oligomer. Target oligomer 10m
M Tris-HCl (PH7.5)-1mM EDTA (PH
8.0). Homogeneity of the completely deprotected oligodeoxyribonucleotides obtained in this way
can be obtained as a single peak in reversed-phase column chromatography, using _T4 -polynucleotide kinase and labeling the 5'-end with [γ- ^32P ]ATP, followed by 15% polyacrylamide gel-7M urea. This was confirmed by the presence of a single band in electrophoresis.

[ka]

[ka]

[ka]

[C] F Transfection into Bm cells Viral DNA of BmNPV T3 strain and pIFN-1
-B241 was adjusted to a molar ratio of 1:100 and mixed with the following composition solution. Distilled water 2.1ml Carrier DNA (salmon testis, 1mg/ml) 50μ BmNPV DNA 10μ pIFN-1-B241 DNA 50μg 2M calcium chloride solution 300μg 50mM containing 0.28M sodium chloride
HEPES buffer (PH7.1) 2.5ml Phosphate buffer (35mM Na ₂ HPO ₄ -35mM
NaH ₂ PO ₄ ) 50μ 1 ml of the resulting suspension was added to 4 ml of Bm cell culture medium, and the above DNA was introduced into silkworm cells.
After 20 hours, the medium was replaced with fresh medium, and after further culturing for 5 days, the medium was collected. The supernatant obtained by centrifugation and clarification was collected and used for α-IFN activity assay. G. Expression of α-IFN in silkworms 0.5 ml/animal (10 ⁷ pfu) of the culture medium described in Section F after 5 days of culture was injected subcutaneously into 5th instar and 1 day old silkworms, and incubated at 25°C for 5 days. , after being reared with mulberry leaves,
A collection needle was inserted into the abdominal leg, the body fluid was collected into an ice-cold Etzpendorf tube, an equal volume of TC-10 medium was added, centrifuged (10,000 rpm, 5 minutes), and the supernatant was collected for α-IFN activity assay. It was used for. H α-IFN bioassay (i) Activity measurement Report by C. Philip et al. (Method in
FL cells (derived from human amniotic membrane), VSV
(vesicular stomatitis virus) in a 96-well microtiter tray.
50% CPE inhibition assay (cytopathic effect inhibition assay)
It was measured by The results are shown in Table 1.

【table】

[Table] Although we applied for depositing the BmNPV and Bm cells used in the present invention to the Institute of Microbial Technology, the deposit was refused. The experiments of the present invention were conducted based on the recombinant DNA experiment guidelines and the recombinant DNA experiment plan submitted to the Science and Technology Agency.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of an example of plasmid operation useful for producing recombinant BmNPV DNA.

Claims (1)

[Claims] 1. Bombyx mori nuclear polyhedrosis virus (BmNPV)
Polyhedral protein structural gene translation initiation codon of DNA
The 5′ upstream DNA fragment containing the promoter that exists between ATG and the Hind site approximately 4.5 Kbp upstream thereof (), the translation start codon (), the structural gene for producing peptides, proteins, or glycoproteins (), and the translation stop codon () BmNPV or BmNPV DNA recombined with double-stranded DNA containing the 3′ downstream DNA fragment () of the polyhedral protein structural gene of BmNPV DNA in cultured silkworm cells or in living silkworms. 1. A method for producing a peptide, protein, or glycoprotein, the method comprising propagating the peptide, protein, or glycoprotein. 2. The method for producing a peptide, protein, or glycoprotein according to claim 1, wherein BmNPV or BmNPV DNA is propagated in living silkworms.