JPH07289270A - Vector for producing peptides - Google Patents

Abstract

PURPOSE:To obtain a vector, capable of providing a high production amount and expressible with a eucaryotic cell in a method for expressing a desired protein using a recombinant DNA technique. CONSTITUTION:(1) This vector is a recombinant Mombyx mori nuclear polyhedrosis virus DNA (BmNPV DNA) having a base sequence of a type in which all or a part of a polyhedral protein gene in the BmNPV DNA is joined through or not through a direct bond base sequence to a desired protein structural gene in the polyhedral protein structural gene part of the BmNPV DNA. (2) This plasmid has a 5'-upstream base sequence containing a promoter part of the BmNPV DNA, followed by all or a part of the polyhedral protein structural gene and further the desired protein structural gene and a 3'-downstream base sequence. (3) This method for producing a recombinant nuclear polyhedrosis virus is to recombine the plasmid with the BmNPV and prepare the virus.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a recombinant DNA virus vector for peptide production.

[0002]

2. Description of the Related Art A method for producing peptides using silkworm nuclear polyhedrosis virus (BmNPV) has already been reported (References 1-3. Shown).

The center of the method is to produce recombinant BmNPV of the type in which the structural gene portion of the polyhedron protein is replaced with the structural gene of the desired protein by the recombinant DNA technique, and the recombinant BmNPV is propagated in silkworm cultured cells or silkworm living organisms. To produce the target protein.

However, this method is not sufficient to produce the desired protein. Therefore, the present inventor has conducted research to develop a more excellent method and completed the present invention.

Although the production of the fusion protein has already been carried out using Escherichia coli, it cannot be said that the yield is significantly improved (References 4 to 10).

[0006]

[Means for Solving Problems]

(1) The present invention relates to silkworm nuclear polyhedrosis virus DNA (BmNPV
() DNA polyhedron protein structural gene part, () all or part of that gene, and () the structural gene of the desired protein is connected by () with or without a bond base sequence. A recombinant silkworm nuclear polyhedrosis virus having

(2) The present invention comprises 5'containing the () promoter portion of silkworm nuclear polyhedrosis virus DNA (BmNPV DNA).
There may be all or part of the () polyhedron protein structural gene following the upstream nucleotide sequence, and then there may be () bond nucleotide sequence, and () the structural gene of the desired protein (() A plasmid having a 3'downstream base sequence () which may or may not contain a terminator) and () does or does not contain a terminator sequence of silkworm nuclear polyhedrosis virus DNA.

(3) The present invention comprises 5'containing the () promoter portion of silkworm nuclear polyhedrosis virus DNA (BmNPV DNA).
There may be all or part of the () polyhedron protein structural gene following the upstream nucleotide sequence, and then there may be () bond nucleotide sequence, and () the structural gene of the desired protein (() It is possible to mix-infect a silkworm culture cell or a silkworm with BmNPV DNA and a plasmid having a 3'downstream nucleotide sequence () which contains a terminator) and () does not contain a terminator sequence of silkworm nuclear polyhedrosis virus DNA (). Characterized recombinant BmNP
It relates to a method for producing V DNA.

Although BmNPV is widely known to sericulture farmers,
A typical strain isolated by the present inventor is T3 strain, and the viral DNA (BmNPV DNA) of this strain is ATCC No.
It has been deposited as 40188.

To extract the portion containing the polyhedrin protein structural gene from this viral DNA, use the method described in References 1-3.
EcoRI treatment is appropriate, and this EcoRI-EcoRI fragment (about
10.5 kb) was introduced at the EcoRI cleavage point of pBR322.
E. coli harboring pBmE36 (E. coli K12 JM83 DGB-0036)
Deposited as FERM BP-813 at Institute of Microbial Technology.

Further, as described in Reference 1, pBmE36
Treated with Hind III to contain polyhedrin protein structural gene
Take the 3.9 kb fragment and use it as a commercially available plasmid pUC9 (Pha
rmacia PL Biochemical), digested with EcoRI, treated with Bal31 (fragments of various lengths can be produced by adjusting the treatment time), and further digested with Hind III. Fragments having all or part can be produced. To connect the structural gene of the desired protein to all or part of the polyhedrin protein gene, an appropriate bond (DNA) can be used, and it corresponds to this bond base sequence in the produced fusion protein. Cleavage of the translated peptide or amino acid (connecting portion) by an appropriate means produces a desired protein, which can be isolated by a conventional method. In the case where the fusion protein itself is useful, the cleavage operation is unnecessary and the bond may not be necessary.

[0012] Peptides or amino acids corresponding to a bond (linkage part) and their cleavage / decomposition methods include Ile-Glu-Gl.
Blood coagulation factor Xa (abbreviated as F-Xa) that cleaves the y-Arg sequence, collagenase that cleaves the amino acid sequence of Pro-Ama-Gly-Pro (Ama is any amino acid) between Ama and Gly, Arg
Or trypsin, Tyr or Phe, which cleaves after Phe
Well-known are chymotrypsin, which cleaves after, S thrombin, which cleaves after Pro-Arg, N-bromosuccinimide, which decomposes tryptophan, and cyanogen bromide, which decomposes methionine (References 4 to 13).

The connecting part and its cleavage / decomposition method are not necessarily limited to these, and various peptides or amino acids may be used in combination with a chemical or enzymatic method.

Desirably, the desired protein does not contain a peptide or amino acid to be decomposed by the above-mentioned method of cleaving / decomposing a connecting portion. However, it is also possible to achieve the purpose by appropriately setting mild conditions and limiting decomposition.

The desired protein means various physiologically active substances, diagnostic reagent substances, industrially useful enzymes and the like, food additives and feed additives, such as interferon (IF
N), tumor necrosis factor (TNF), lymphokines such as interleukins, hormones such as insulin and growth hormone, various antigenic proteins such as hepatitis vaccine and influenza vaccine, and tissue plasminogen activator (TP)
Enzymes such as A), somatomedin, acylase, lipase, amylase and steroid converting enzyme can be mentioned.

[0016] Depending on the substance, sugar chains and the like may be added to these substances, but in the method of the present invention for producing peptides in a eukaryotic cell, when a eukaryotic gene is used, it will be in vivo. Since it undergoes various modifications similar to those described above, it can be said that it is more useful than the method using bacteria.

The genes encoding these proteins may be either cDNA produced through chromosomal DNA or mRNA extracted from natural raw materials, chemically synthesized DNA, or those produced by combining these means.

Further, the target substance may be modified intentionally even if some differences (substitution, loss, addition) occur in the amino acid sequence, as long as the required functions such as physiological activity can be obtained. It may be one. For example, when the methionine is to be decomposed with cyanogen bromide in relation to the above-mentioned connecting portion, it is considered that methionine in the desired protein is replaced with another amino acid or removed. Whether or not the activity of the peptides produced based on the above is suitable for the purpose may be examined.

Further, a part of the amino acid may remain in the desired protein, which may be caused by the chemical decomposition of cysteine with 2-nitro-5-thiocyanobenzoic acid, but in this case as well. The activity may be examined to determine whether it suits the purpose.

Silkworm nuclear polyhedrosis virus DNA (BmNPV DNA)
A plasmid in which all or part of the polyhedrin protein gene of (1) and a structural gene of the desired protein are linked (with or without a bond base sequence), that is, a fusion protein gene is incorporated. The production of the replacement plasmid)
Normal DNA using commercially available plasmids and restriction enzymes
This can be done by operation, and can be easily performed by referring to the examples described later.

To produce a recombinant virus using this, for example, cultured silkworm cells are treated with a recombinant plasmid and BmNPV.
Mixed infection with and, if necessary, plaque method (Reference 14)
The recombinant virus is isolated by the dilution method (Reference 15) or the like.

Bm cells (BM-N cells of References 1, 14 and 16: ATCC No. CRL89) are used as silkworm culture cells.
10) and BM-N cells deposited as ATCC No. CRL-8851 are known. To produce the target protein using silkworm, the virus (mixture of recombinant virus and non-recombinant virus or isolated recombinant virus) grown in cultured cells is transdermally transferred to silkworm. Alternatively, inject it into the body cavity to infect it, or infect it by mixing the virus with artificial feed or mulberry leaf to infect it, raise it on artificial feed or mulberry leaf for an appropriate number of days, accumulate the desired fusion protein, and collect the body fluid. The precipitate is collected by centrifugation, etc., or the silkworms are ground and crushed, and sodium dodecyl sulfate (SDS) aqueous solution,
It can be obtained by extraction with a urea aqueous solution or an alkaline aqueous solution and a conventional treatment. When cleaving the fusion protein, the cleaving / decomposing operation may be performed as described above.

Examples of desired proteins such as insulin-like growth factor (IGF-I, IGF-II) and interferon (α-IFN) will be described below, but these are merely examples and have a limited meaning. There is no. The outline of plasmid construction is shown in FIGS. 15 to 20. By convention, the left side is shown as 5 '(upstream) and the right side is shown as 3' (downstream), except as otherwise noted.

For synthesis, cleavage, screening, isolation, etc. of DNA fragments such as polyhedron protein gene, desired protein gene and fusion protein gene, refer to References 1 to 3 and general gene manipulation techniques (see, for example, References 21 and 22). It can be applied.

[0025]

INDUSTRIAL APPLICABILITY The recombinant silkworm nuclear polyhedrosis virus according to the present invention is composed of a fusion gene in which a part of the silkworm nuclear polyhedrosis gene sequence is fused with the sequence of the structural gene of the protein desired to be produced. The recombinant silkworm nuclear polyhedrosis virus is characterized by being capable of producing a desired protein by using the recombinant silkworm nuclear polyhedrosis virus as a vector.

That is, recombinant silkworm nuclear polyhedrosis virus in which a structural gene of a protein desired to be produced was introduced into a prescribed site of silkworm virus (BmNPV) was infected into a silkworm cultured cell or a silkworm living body, and was infected for a certain period of time. To produce the desired protein in the living body of the silkworm by culturing the cells in the cultured cells or extracellular fluid, or in the case of infecting the living body of the silkworm, by raising the infected silkworm for a certain period of time. You can The produced protein has a remarkably large amount of production as compared with the case where the fusion gene vector is not used, and further, the protein is modified with sugar chains peculiar to eukaryotic cells.

Furthermore, the plasmid according to the present invention has utility as an intermediate material for producing a recombinant silkworm nuclear polyhedrosis virus capable of producing the above-mentioned useful proteins. According to the vector production method of the present invention, the silkworm recombinant viral DNA can be produced.

[0028]

EXAMPLES Example 1 A. Construction of a plasmid from which the polyhedron gene has been removed Plasmid pBmE36 (References 1 to 3) is cleaved with EcoRI and dNTP
s (4 kinds of deoxynucleoside triphosphates)
Blunted with DNA polymerase I (Klenow fragment) in the presence. This was partially digested with HindIII and the fragment containing the polyhedron gene was separated by agarose gel electrophoresis. This was blunt-ended with Klenow fragment after cutting pUC9 with EcoRI, and further ligated with T4 ligase to that cut with HindIII. This was used to transform Escherichia coli K-12 JM83 and the plasmid was collected into p9BmE.
36 (Fig. 15).

The sites recognized by EcoRV and AatI at the start codon ATG and the stop codon TAA, respectively, were identified by in vitro mutagenesis (Reference 17).
(Fig. 15). That is, p9BmE36 was treated with DNase I in the presence of ethidium bromide to prepare a nicked plasmid, and the following two kinds of DNA fragments chemically synthesized in advance were annealed (FIG. 1).

Then, this was treated with polymerase I and T4 ligase, Escherichia coli K-12 JM83 was transformed with the resulting heteroduplex plasmid, and again transformed to obtain the desired mutant by transforming plasmid p9BmM3.
6 was obtained (FIG. 15).

The polyhedrin gene was removed from p9BmM36, and the following DNA fragments (38mer two species) were chemically synthesized to insert a polylinker (FIG. 2).

These two fragments are complementary to each other and, after annealing, SacI, SmaI, EcoRI, Nco
Generates sites recognized by I, EcoRV and XbaI.

The above two fragments were annealed after phosphorylating the 5'end with T4 kinase. This is p9
BmM36 was digested with EcoRV and AatI, and then ligated with a fragment excluding the polyhedron gene portion using T4 ligase. This operation newly added BglII to the 5'side and Aa to the 3'side of the linker.
The site recognized by tI occurs. E. coli with this plasmid
K-12 JM83 was transformed to obtain a plasmid, which was designated as pBM030 (Fig. 15).

The DNA sequence and the cleavage point of the polylinker part of pBM030 are shown in correspondence with the polyhedron gene part of p9BmE36 (FIG. 3).

B. Plus with a partially deleted polyhedrin gene
Preparation of amide Bal31 after cutting plasmid p9H18 (References 1-3) with EcoRI
Treated to scrape both sides from the cut point. By varying the treatment time of Bal31, fragments of various lengths were produced. Hind this
Treatment with III, separation and extraction by 0.7% agarose gel electrophoresis, and virus-derived DNA fragments of various lengths were obtained (FIG. 16).

PUC9 was treated with SmaI and HindIII and ligated with the previously obtained DNA fragment (having blunt ends and HindIII ends) to prepare a plasmid, which was used to transform Escherichia coli K-12 JM83. After growing this, take the plasmid,
Primer the nucleotide sequence from the 3'side of the inserted virus-derived DNA fragment (15 base sequencing primer for M13)
Was determined by the dideoxy method (dideoxy method: reference 18) to identify the viral polyhedrin gene portion (FIG. 16). That is, the base sequence corresponding to the amino acid sequence of the polyhedron protein described in Celebrani et al. (Reference 19) was found in the base sequence of a virus-derived DNA fragment, and the translation initiation codon ATG and the termination codon TAA were determined.

Among various plasmids (p9B series plasmids) obtained corresponding to the treatment time of Bal31, ones lacking 212 bp, 338 bp, 662 bp and 727 bp or less downstream of the translation initiation codon ATG of the polyhedron gene were deleted. P9B240 and p9B12 respectively
0, p9B115 and p9B086. The polyhedron gene is 738 bp including the termination codon TAA (Figs. 4 and 16).

C. α-interferon and polyhedron protein
Preparation of gene for fusion protein of pIFN2B310 (References 1 to 3) was digested with SmaI, and α-IFN-J fragment was separated by agarose gel electrophoresis. This was ligated to pBM030 cleaved with Sma I with T4 ligase, and Escherichia coli K-12 JM83 was transformed with the resulting plasmid. The resulting recombinant plasmid had α-I inserted in the correct orientation.
It was confirmed to have the FN-J gene and designated as pBT310 (Fig. 17).

After pBT310 was cleaved with BglII, it was blunt-ended with Klenow fragment in the presence of dNTPs and then cleaved with ScaI. Perform agarose gel electrophoresis and α-IFN-J
The fragment containing the gene was isolated (Figure 17).

P9B086 was cleaved with EcoRI and ScaI, a fragment containing the polyhedron gene was separated by agarose gel electrophoresis, and the fragment obtained by blunting it with S1 nuclease and the previously obtained α-IFN-J gene were separated. The containing fragment was ligated with T4 ligase. The resulting plasmid is E. coli K-12 JM83
Was confirmed by the dideoxy method that the DNA sequence of the binding part of the obtained plasmid was correct, and pIFNF086 was obtained (FIGS. 5 and 17).

D. Of the fusion protein of IGF-I and polyhedron protein
Gene preparation Plasmid from E. coli K12 MC1061 IGF001 (FERM BP-932)
pIGF001 was collected, digested with SalI, blunt-ended with Klenow fragment in the presence of dNTPs, digested with AvaII, and subjected to polyacrylamide gel electrophoresis to isolate a fragment containing the IGF-I gene (FIG. 18).

Separately, pBM030 was cut with EcoRV and SacI (FIG. 18).

When a fusion protein of a polyhedron protein and IGF-I is made so that the fusion protein can be recognized and cleaved by the blood coagulation factor Xa (F-Xa), an Ile-Glu-Gly-Arg is formed between the two. It was decided to introduce the amino acid sequence of For that purpose, the following two DNA fragments were chemically synthesized (Fig. 6).

These two fragments are complementary to each other and, after annealing, give rise on the upstream side a terminus capable of binding the SacI breakpoint and on the downstream side a cohesive terminus capable of binding the AvaII breakpoint.

These two fragments were annealed after phosphorylating the 5'end with T4 kinase and then annealing.
The FI fragment and the cleaved pBM030 were ligated using T4 ligase. E. coli K-12 JM83 was transformed with this to obtain a recombinant plasmid. This plasmid pIG
F-I030 was confirmed to have correctly bound the F-Xa recognition site and the IGF-I gene by determining the DNA sequence by the dideoxy method. pIGF-I030 was digested with BglII, blunt-ended with Klenow fragment in the presence of dNTPs, digested with ScaI, and the fragment containing IGF-I gene was separated by agarose gel electrophoresis (FIG. 18).

Separately, p9B086 was digested with EcoRI and ScaI to give S1.
The fragment containing the polyhedron gene was separated by agarose gel electrophoresis with blunt ends using nuclease, and the fragment containing the IGF-I gene was ligated with T4 ligase. Escherichia coli K-12 JM83 was transformed with the resulting plasmid to obtain a plasmid pIGF-IF086 having the fusion protein gene correctly bound (Fig. 18). DNA at the binding site
The sequence is shown (Fig. 7).

E. IGF-II and polyhedron proteins of various lengths
Preparation of fusion protein gene with E. coli K12 MC1061 IGF002
The plasmid pIGF002 was collected from (FERM BP-933) and the SalI
After partial digestion with, Klenow fragment in the presence of dNTPs was used to make a blunt end, which was then cleaved with HaeIII was subjected to polyacrylamide gel electrophoresis to separate the fragment containing the IGF-II gene. Add this to pBM030 with SmaI
Cleavage with T4 ligase and ligation
K-12 JM83 was transformed. The resulting transformant is IGF-I
Plasmid pIGF with the I gene inserted in the correct orientation
-Confirmed to have II 030 (Fig. 19).

PIGF-II 030 was partially digested with EcoRI, cut with ScaI, and the fragment containing the IGF-II gene was separated by agarose gel electrophoresis. This was ligated with T4 ligase to a fragment containing a part of the polyhedron gene separated by agarose gel electrophoresis after cutting p9B240, p9B120 or p9B115 with EcoRI and ScaI (FIG. 19).

Escherichia coli K-12 JM83 was transformed with the resulting plasmid, and it was confirmed by the dideoxy method that the plasmid contained in the obtained transformant had the gene of the fusion protein bound correctly. .

Each of these plasmids was designated as pIGF-II F.
240, pIGF-II F120 or pIGF-II F115 (Fig. 1
9).

The DNA sequence of the binding portion of each plasmid is shown (FIG. 8).

Example 2: Transfection of Bm cells
Virus DNA of emissions BmNPV T3 strain (ATCC N0.40188) and pIFN F08
The following composition liquid I was prepared by adjusting the molar ratio of 6 to 1: 100.
And II were mixed.

I. Distilled water 2.1 ml Carrier DNA (salmon testis, 1 mg / ml) 50 μl BmNPV DNA (1 mg / ml) 10 μl pIFN F086 DNA 50 μg 2M calcium chloride solution 300 μg II. 50 mM HEPES buffer containing 0.28 M sodium chloride (pH 7.1) 2.5 ml Phosphate buffer (35 mM Na ₂ HPO ₄ -35 mM NaH ₂ PO ₄ ) 50 μl

1 ml of the resulting suspension was added to Bm cell culture medium (1
4 ml of TC-10 medium (Reference 20) containing 0% fetal bovine serum was added, and Bm cells (ATCC No. CRL 8910: 2 × 10 ⁶ cells) were cultured (5 ml of medium remained). The above DNA was introduced into Bm cells by adding it to the incubator. 20
After a lapse of time, the medium was replaced with a fresh medium, and the medium was recovered after culturing for 5 days. This was centrifuged, the supernatant was taken, and the cloned recombinant virus was collected by the plaque assay method (Reference 14), which was designated as vIFN F086.

Similarly, pIGF-I F086 and pIGF-II F24
0, recombinant virus from pIGF-II F120 and pIGF-II F115 v
IGF-IF086, vIGF-II F240, vIGF-II F120 and vIGF-I
I got F 115.

Example 3 Expression of Fusion Protein in Bm Cells Bm cells (ATCC No. CRL 8910: 2 × 10 ⁶ cells) were infected with vIFNF086 suspension (5 × 10 ⁷ pfu), and infected for 30 minutes. After that, the supernatant was removed, 4.5 ml of fresh medium was added, and the mixture was cultured at 25 ° C. for 4 days, and then the cultures were collected and centrifuged (1,000 rpm, 10 minutes) to collect the precipitate. To this, 500 μl of fresh medium was added, and freeze-thawing was repeated 5 times, followed by centrifugation (15,000 rpm, 10 minutes) to collect a precipitate. 4% sodium dodecyl sulfate (SDS) -1
200 μl of 0% mercaptoethanol was added and dissolved, and 5 μl of the solution was used as a sample for SDS-14% acrylamide gel electrophoresis.

After electrophoresis, a band corresponding to the fusion protein was observed on the gel. The density was measured with a densitometer and compared with the density of the marker (containing 1 μg of protein in each band) to determine the production amount. It was 0.3 mg per 1 ml of the initial cell culture medium (IFN portion of this fusion protein accounted for about 40% of the calculated molecular weight, so if IFN was separated, 0.12 mg / ml was produced. Has been done). 5x10 ⁶ U / ml (this is 0.005 mg)
/ ml), which is more than 20 times.

Similarly, vIGF-I F086, vIGF-II F24
The amount of the fusion protein produced by Bm cells infected with 0, vIGF-II F120 and vIGF-II F115 was measured and was 0.5 mg, 0.1 mg, 0.4 mg and 0.5 mg per 1 ml of each cell culture medium ( Included in these fusion proteins calculated from molecular weight
The amount of IGF-I or IGF-II is 0.1mg / ml, 0.05mg / ml, 0.1m
g / ml and 0.1 mg / ml).

Example 4: Expression of fusion protein in Bm cells and
BIG cells cleaved with cyanogen bromide (BrCN) (2 × 10 ⁶ cells) were treated with vIGF-II F120 suspension (5 × 10 ⁷ pf).
After 30 minutes, the virus was removed, 4.5 ml of fresh medium was added, the mixture was cultured at 25 ° C. for 4 days, and the culture was collected and centrifuged (1,000 rpm, 10 minutes) to collect the precipitate. To this, 500 μl of fresh medium was added, and freeze-thawing was repeated 5 times, followed by centrifugation (15,000 rpm, 10 minutes) to collect a precipitate. After washing this precipitate with distilled water and dissolving it with 0.5% SDS, high performance liquid chromatography (TSK gel phenyl 5PWRP (Toyo Soda Co., Ltd.) solvent system: 0.1% trifluoroacetic acid-acetonitrile 20% to 75% linear concentration Gradient) containing fusion protein
A 2 ml fraction was collected. To this, add 50 μl of 1% SDS, concentrate to dryness, and dissolve in 50 μl of distilled water.
Take 15 μl and add 35 μl formic acid and 45 μl 70% formic acid. Add 5 μ of 70% formic acid solution of cyanogen bromide (200 μmol / ml).
was added, and the mixture was reacted at room temperature for 24 hours, concentrated to dryness, and added to 7.5 μl.
When it was dissolved in distilled water of No. 1 and electrophoresed, a band corresponding to IGF-II could be detected.

That is, in electrophoresis using SDS-16% polyacrylamide gel, Bm infected with vIGF-II F120 was analyzed.
In the fraction extracted from cells with SDS aqueous solution and partially purified by high performance liquid chromatography, the band corresponding to the fusion protein of polyhedral protein (partial) and IGF-II was about 23K.
In addition, when the product was decomposed with cyanogen bromide, a band of about 7K corresponding to IGF-II and several bands of 10 to 14K which were presumed to be decomposition products of polyhedron protein were observed.

Determination of the amino terminus of IGF-II Bm infected with vIGF-II F120 was prepared as described above.
About 20 mg of the fusion protein was taken from 50 ml cell culture, cleaved with cyanogen bromide, and the reaction solution was concentrated.

This concentrated solution was dissolved in 200 mM pyridine-acetic acid buffer (pH 3.0) and subjected to column chromatography (SP-Toyopearl (Toyo Soda Co., Ltd.) solvent system: 200 mM pyridine-
Fractions containing IGF-II were collected using an acetic acid buffer (pH 3.0) to a 2.0 M pyridine-acetic acid buffer (pH 5.0) linear concentration gradient). After freeze-drying this fraction, dissolve it in distilled water and perform high performance liquid chromatography (OD
IGF-II was obtained by purification with S-120T (Toyo Soda Co., Ltd.) solvent system: 0.1% trifluoroacetic acid-acetonitrile 10% to 65% linear concentration gradient. Peptide sequencer (470A
The amino acid sequence on the amino terminal side of this IGF-II was examined by a protein sequencer (Applied Biosystems). The confirmed sequences are shown below (Fig. 9).

Example 5: Production of fusion protein by Bm cells
The plasmid pIGF001 was collected from Escherichia coli K12 MC1061 IGF001 (FERM BP-932) cleaved with collagenase . This was cleaved with SalI, blunt-ended with Klenow fragment in the presence of dNTPs, and cleaved with Ava II, and subjected to polyacrylamide gel electrophoresis to isolate a fragment containing the IGF-I gene (Fig. 2).
0).

Separately, pIGF-II F120 was digested with EcoRV and EcoRI to remove the IGF-II gene (FIG. 20).

[0065] The polyhedron protein and the IGF-probe so that the collagenase that cleaves the amino acid sequence of Pro-Ama-Gly-Pro (Ama is an arbitrary amino acid) between Ama and Gly can be recognized and cleaved.
When making a fusion protein with II, Gly-Pro-Ala-Gly-
It was decided to introduce the amino acid sequence of Pro-Ala. For that purpose, the following two DNA fragments were chemically synthesized (Fig. 1
1).

These two fragments are complementary to each other, and after annealing, an end capable of binding to the EcoRI breakpoint is formed on the upstream side, and a cohesive end capable of binding to the Ava II breakpoint is generated on the downstream side.

These two fragments were annealed after phosphorylating the 5'end with T4 kinase, and the above fragments of IGF-I and cleaved pIGF-II F.
120 was ligated with T4 ligase. E. coli K-12 JM83 was transformed with this to obtain a recombinant plasmid (FIG. 20). In this plasmid pIGF-IF120, it was confirmed that the collagenase recognition site and the IGF-I gene were correctly bound by determining the DNA sequence by the dideoxy method.

The DNA sequence of the binding portion and the corresponding amino acid are shown (FIG. 11).

Similar to the method described in Example 2, pIGF-I
Recombinant virus vIGF-IF120 was obtained using F120.

Similar to the method described in Example 4, vIGF-IF1
Bm cells infected with 20 were collected from 50 ml of culture, freeze-thawed, centrifuged (15,000 rpm, 10 minutes), and the precipitate was collected. This was extracted with 6M guanidine hydrochloride and centrifuged (15,
(000 rpm, 5 minutes) After removing the residue, dialysis was performed and the resulting precipitate was collected by centrifugation (15,000 rpm, 10 minutes).

This was dissolved in 800 μl of 10 M urea to obtain 200 mM.
Calcium chloride 100 μl, 250 mM Tris-HCl (pH 7.4) 2
00 μl was added. Add collagenase solution (1,500 U / m
l: manufactured by Sigma Chemical Co., Ltd.) (900 μl) was added, and the mixture was reacted at 30 ° C. for 2 hours and then centrifuged (15,000 rpm, 5 minutes) to collect a supernatant. This was subjected to ion exchange chromatography (DEAE-Toyopearl (Toyo Soda Co., Ltd.)) solvent system: 25 mM Tris-hydrochloric acid (pH 7.4), and IG having an extra amino acid sequence at the N-terminal
The fraction containing FI (abbreviated as IGF-IP) was taken.
After concentrating this, high performance liquid chromatography (RPSC (Beckman) solvent system: 0.1% trifluoroacetic acid-acetonitrile
IGF-IP was purified by 10% -65% linear gradient).
When the N-terminal amino acid sequence of this IGF-IP was examined using a 470A protein sequencer (Applied Biosystems), it was found that the fusion protein was cleaved at the correct position by collagenase.

The amino acid sequence on the amino terminal side of the confirmed IGF-IP is as follows (FIG. 12).

Example 6: Polyhedra in silkworm individuals-IGF
-I fusion protein expression Infused silkworms on the 5th day, 1st day, with vIGF-IF086 suspension 0.5 ml / mouse (10 ⁷ pfu) percutaneously and raised with mulberry leaves at 25 ° C for 3 days. Then, insert the sampling needle into the abdominal leg to collect the body fluid in an ice-cold Eppendorf tube, and centrifuge (15,000 rpm,
10 minutes) and a precipitate was collected. To this, add 200 μl of 4% SDS-10% mercaptoethanol and dissolve.
Was used as a sample for SDS-14% acrylamide gel electrophoresis. A band corresponding to the fusion protein was observed in the gel after electrophoresis, so the density was measured with a densitometer, and the amount of production was measured by comparison with the density of the marker (containing 1 μg of protein in each band). , 5 mg per 1 ml body fluid
(If IGF-I was separated from this fusion protein, the calculated molecular weight would be 1 mg / ml).

In electrophoresis using SDS-14% polyacrylamide gel, BmNPV-T3 strain-infected silkworm body fluid showed a clear band corresponding to polyhedrin protein at about 30 K, whereas vIGF
-IF086 Infected silkworm body fluid did not have a band corresponding to polyhedron protein, but a characteristic band of a fusion protein of polyhedron protein and IGF-I was observed at about 36K. IGF-I used in the examples and
The DNA sequence and amino acid sequence of the synthetic gene of IGF-II are shown below (FIGS. 13 to 20).

Document 1: Nature 315 592 (1985) Document 2: Japanese Patent Laid-Open No. 61-9288 Document 3: Japanese Patent Laid-Open No. 61-9297 Document 4: Science 198 1056 (19)
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[Brief description of drawings]

FIG. 1 DNA sequences of two fragments used for in vitro mutagenesis.

FIG. 2 DNA sequence of a fragment for inserting a polylinker

FIG. 3: DNA sequence and cleavage point of polylinker part of pBM030

FIG. 4 DNA sequence of a plasmid having a partially deleted polyhedrin gene

FIG. 5: DNA sequence of the binding part of pIFN086

FIG. 6 DNA sequence of a fragment for introducing an F-Xa recognition amino acid sequence

FIG. 7: DNA sequence of the binding site of pIGF-IF086

FIG. 8: DNA sequence of each plasmid binding site

FIG. 9: Amino acid sequence on the amino terminal side of IGF-II

FIG. 10: Sequence of DNA fragment for introducing collagenase-recognizing amino acid sequence

FIG. 11: DNA sequence of pIGF-IF120 binding site and corresponding amino acid sequence

FIG. 12: Amino acid sequence on the amino terminal side of IGF-IP

FIG. 13: DNA sequence of IGF-I synthetic gene and corresponding amino acid sequence

FIG. 14: DNA sequence of IGF-II synthetic gene and corresponding amino acid sequence

FIG. 15: Outline of plasmid preparation example for recombinant virus production

FIG. 16: Outline of example of preparation of plasmid for recombinant virus production

FIG. 17: Outline of plasmid preparation example for recombinant virus production

FIG. 18: Outline of plasmid preparation example for recombinant virus production

FIG. 19: Outline of plasmid preparation example for recombinant virus production

FIG. 20: Outline of preparation example of plasmid for recombinant virus production

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:91) (72) Inventor Tadashi Horiuchi 1-16-13 Kitakasai, Edogawa-ku, Tokyo Central Pharmaceutical Research Laboratory (72) Inventor Yoshio Sato 1-16-13 Kitakasai Nishi, Edogawa-ku, Tokyo Daiichi Pharmaceutical Central Research Laboratory (72) Inventor Yoshiyuki Saeki 1-16-13 Kitakasai, Edogawa-ku, Tokyo No. 1 Central Research Institute for Pharmaceutical Sciences

Claims (3)

[Claims] 1. A silkworm nuclear polyhedrosis virus DNA (BmNPV DN
A) A nucleotide sequence of the polyhedron protein structural gene part, in which () all or part of the gene and () the structural gene of the desired protein are linked () with or without a bond base sequence. Recombinant silkworm nuclear polyhedrosis virus having 2. Silkworm nuclear polyhedrosis virus DNA (BmNPV DN
There may be a whole or part of () polyhedron protein structural gene following the 5'upstream nucleotide sequence containing the () promoter portion of (A), and then there may be () bond hand nucleotide sequence. Having the structural gene (() may contain a terminator of the protein) and () the 3'downstream nucleotide sequence containing or not () the terminator sequence of the silkworm nuclear polyhedrosis virus DNA 3. The silkworm nuclear polyhedrosis virus DNA (BmNPV DN
There may be a whole or part of () polyhedron protein structural gene following the 5'upstream nucleotide sequence containing the () promoter portion of (A), and then there may be () bond hand nucleotide sequence. BmNPV DNA with a structural gene of the protein of () (which may include a terminator of the protein) and () with or without the terminator sequence of silkworm nuclear polyhedrosis virus DNA () 3'downstream base sequence A method for producing recombinant BmNPV DNA, which comprises infecting silkworm cultured cells or silkworm mixedly