JP2004254681A - Transgenic vector to insect and method for producing gene product - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a gene engineering material for easily purifying an objective protein without requiring use of recombinant baculovirus, and provide a method for producing a foreign protein by using the gene engineering material. <P>SOLUTION: A large amount of the foreign protein is produced in silk gland cells, outside of silk gland cells or brought to produce silk by introducing an expression gene cassette prepared by fusing a DNA sequence of 5' terminal and a DNA sequence of 3' terminal of fibroin H chain gene with a foreign protein gene. By the new method, foreign protein easily purified is produced using the silk gland without using the recombinant baclovirus, and a technique for producing the foreign protein is established. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a vector for introducing a gene into an insect cell, and a method for producing an exogenous protein using an insect cell, an insect tissue, or an insect transfected using the vector. Furthermore, the present invention relates to a silk thread containing a foreign protein produced by the recombinant silkworm obtained by the present invention.

  Production of foreign proteins using gene recombination techniques is used in various industries. Escherichia coli, yeast, animal cells, plant cells, insect cells and the like are mainly used as the host. However, a host capable of efficiently producing any foreign protein has not been developed, and it is necessary to construct a production system for each target protein, and further technological innovation in foreign protein production technology in each host is desired. ing.

  Bacterial or yeast systems, such as E. coli, have problems with post-translational modifications and may not be able to synthesize proteins in a fully functional manner. In addition, animal cells can often synthesize proteins in a functional form, but are generally difficult to grow and have low yields and are not economical.

  On the other hand, in the production of recombinant proteins using insects or insect cells, it is known that enzymes and useful proteins having biological activity can be mass-produced relatively inexpensively, and post-translational modification of proteins similar to animals can be obtained. I have. Specifically, by infecting insects or insect cells with a baculovirus incorporating a foreign protein gene, foreign proteins can be mass-produced relatively inexpensively, and bioactive proteins commercialized as pharmaceuticals are also known. (JP-A-61-9288, JP-A-61-9297).

  However, conventional recombinant protein production technology using insects or insect cells uses a recombinant virus to introduce a foreign gene, and thus requires inactivation and containment for safety reasons. There is. In addition, in the method of inoculating a silkworm with a recombinant virus, the inoculation of the recombinant virus is complicated, and the target foreign protein is produced in the body fluid. It was necessary to purify from the protein. For this reason, there was a problem that it was difficult to obtain a highly pure recombinant protein.

On the other hand, in recent years, recombination of foreign genes into insect chromosomes has been attempted, using the DNA of Autographa californica nuclear polyhedrosis virus (AcNPV), a kind of nucleopolynucleosis virus, to jellyfish green fluorescent protein in the silkworm fibroin L chain gene. A method has been developed in which the gene-added fusion gene is introduced into the silkworm chromosome by homologous recombination and expressed (Genes Dev., 13 , 511-516, 1999), and the human collagen gene is introduced using this technique. The disclosed silkworm and its manufacturing method are disclosed (JP-A-2001-161214).

Recently, a method of stably introducing a foreign gene into a silkworm chromosome using piggyBac, a transposon derived from a lepidopteran insect, and expressing a protein encoded by the foreign gene has been studied using a jellyfish green fluorescent protein as a model. It was also confirmed that the gene was stably transmitted to offspring by the cross (Nature Biotechnology 18 , 81-84, 2000).

  However, in the above-described method for introducing a foreign protein gene into insect chromosomes using AcNPV, since a recombinant baculovirus (AcNPV) is used, the problem of inactivating or containing the recombinant virus still remains. Next, in the example using the transposon piggyBac, since the amount of green fluorescent protein produced is not sufficient and the whole silkworm is produced, the expressed recombinant green fluorescent protein is recovered in a highly pure form. In order to achieve this, there is an economical problem because advanced purification technology is required.

  In other words, in the technology for producing a foreign protein using such insect cells as a host, it is necessary to inactivate or contain the recombinant baculovirus, or to obtain a target protein from a body fluid containing a large amount of contaminating proteins such as when using silkworms. There are several problems such as difficulty in purifying the protein and low expression of the target protein.

JP-A-61-9288 Japanese Patent Publication No. 61-9297 JP 2001-161214 Public Relations

Genes Dev., 13,511-516,1999 Nature Biotechnology 18,81-84,2000

  Although recombinant protein production technology using insects has been studied energetically, it is necessary to inactivate and contain the recombinant baculovirus incorporating the foreign protein gene. There are problems such as complexity. In addition, in the production of foreign proteins in silkworms using a recombinant baculovirus, there was a problem that it was difficult to extract and purify the target protein from a body fluid containing a large amount of contaminants.

Studies have been conducted on the production technology of recombinant proteins that introduce foreign protein genes into the silkworm chromosome, but the production of the foreign protein of interest is low, and it is difficult to purify the protein of interest from silkworm body fluids. There are also issues.
In view of such circumstances, the present invention provides a genetically engineered material for insects that does not require the use of a recombinant baculovirus and can facilitate purification of a target protein, and at the same time, utilizes the genetically engineered material. It is an object to provide a method for producing a foreign protein.

  The present inventors have focused on the fact that silkworm silk glands, particularly posterior silk glands, produce a large amount of fibroin, which accounts for 70 to 80% of the silk protein, and secrete them out of the cells of the silk gland. A gene cassette in which the 5 'end of the foreign protein gene is connected to the 3' end of the 5 'end containing the first intron of the fibroin H chain gene downstream of the promoter expressed in the gland so that the amino acid frame is continuous. It has been found that introduction of the protein into silk gland cells dramatically improves the amount of foreign protein produced.

  When a fusion gene in which the 3 ′ end of the fibroin H chain gene is linked to the 3 ′ end of the foreign protein gene so that the amino acid frame is continuous is expressed under the control of a promoter that expresses in the silk gland, the foreign protein Is secreted and produced in large quantities outside the silk gland cells. In addition, the DNA sequence of the 5′-terminal portion including the first intron of the fibroin H chain gene on the 5 ′ side of the foreign protein gene, the DNA sequence of the 3 ′ terminal portion of the fibroin H chain gene on the 3 ′ side, and the amino acid frame, respectively. A gene cassette designed to be a continuum was created, and a recombinant silkworm in which this gene cassette was introduced into the chromosome was produced.The recombinant silkworm produced a large amount of the target protein in the silk thread. Was found.

That is, the present inventors have introduced a gene cassette for expression obtained by fusing the DNA sequence of the 5 ′ end portion and the DNA sequence of the 3 ′ end portion of the fibroin H chain gene to a foreign protein gene into silk gland cells and the like. A large amount of foreign protein was successfully produced in silk gland cells, extracellular silk gland cells, and silk, and purified without using recombinant baculovirus and using the silk gland to produce foreign protein. A foreign protein production technology that facilitates the process has been established.
The present invention relates to a genetic engineering material, a transformant, a method for producing a foreign protein using the transformant, and a silk thread containing the foreign protein, such as the following gene cassette and vector, which can be used for the production of a foreign protein in an insect.

1) (1) a promoter expressed in the silk gland;
(2) a gene obtained by fusing the 5 ′ end of the fibroin H chain gene to the 5 ′ side of the foreign protein structural gene, which is linked downstream of (1);
A gene cassette for expression of a foreign protein, comprising:

2) (1) a promoter expressed in the silk gland;
(2) a gene obtained by fusing the 3 ′ end of a fibroin H chain gene to the 3 ′ side of a foreign protein structural gene not containing a termination codon and connected downstream of the above (1);
Or (1) a promoter expressed in the silk gland; and (2) a 3 'end of the 3' end of the fibroin H chain gene, which is connected downstream of (1). A gene cassette for expression of a foreign protein, comprising: a gene obtained by fusing a foreign protein structural gene.

3) (1) a promoter expressed in the silk gland;
(2) The 5 ′ end of the fibroin H chain gene is fused to the 5 ′ side of the foreign protein structural gene not containing a stop codon and downstream of the above (1), and the fibroin is fused to the 3 ′ side of the structural gene. A gene obtained by fusing the 3 'end of the H chain gene;
A gene cassette for expression of a foreign protein, comprising:

4) The foreign protein according to 1) or 3), wherein the 5 ′ terminal portion of the fibroin H chain gene contains a part of the first exon, the first intron, and the second exon of the fibroin H chain gene. Expression gene cassette.
5) The gene cassette for expression of a foreign protein according to 4), wherein the portion of the first exon and the second exon of the fibroin is a secretory signal gene region of a fibroin H chain gene.

6) (1) the promoter expressed in the silk gland and (2) the 5 ′ end portion of the fibroin H chain gene linked downstream of (1) above are DNAs shown in SEQ ID NO: 1 or SEQ ID NO: 2. The gene cassette for expression of a foreign protein according to 5), which is characterized in that:
7) The gene cassette for expressing a foreign protein according to any one of 1) to 6) above, wherein (1) a silk gland promoter and / or (2) a fibroin H chain gene contained in the gene cassette for expressing a foreign protein. A gene cassette for expression of a foreign protein, wherein one or more bases are added, deleted or substituted with other bases within a range where the 5 ′ terminal portion of the above has substantially the same function.

8) The gene cassette for expressing a foreign protein according to any one of 1) to 7) above, wherein (1) a silk gland promoter portion and / or (2) a fibroin H chain contained in the gene cassette for expressing a foreign protein. For expression of a foreign protein, which has a gene that hybridizes with the portion under stringent conditions in place of the 5′-terminal portion of the gene and has a gene having substantially the same function as the portion. Gene cassette.
9) The gene cassette for expressing a foreign protein according to 2) or 3), wherein the 3′-terminal portion of the fibroin H chain gene contains at least one codon encoding cysteine.

10) The gene cassette for expressing a foreign protein according to any one of 2), 3) and 9), wherein the 3 ′ terminal portion of the fibroin H chain gene is the DNA shown in SEQ ID NO: 3.
11) In the gene cassette for expression of a foreign protein according to 10), addition of one or several bases to the extent that the 3 ′ end of the fibroin H chain gene has substantially the same function, A gene cassette for expression of a foreign protein, which has a deletion or substitution to another base.

12) The gene cassette for expression of a foreign protein according to 10) or 11), wherein the 3 ′ end of the fibroin H chain gene is replaced with the 3 ′ end of the fibroin H chain gene under stringent conditions. A gene cassette for expression of a foreign protein, characterized in that the gene cassette has a gene that hybridizes with and has substantially the same function.
13) The promoter expressed in the silk gland is at least one promoter selected from a promoter of a fibroin H chain gene, a promoter of a fibroin L chain gene and a promoter of a sericin gene. A gene cassette for expressing a foreign protein according to any one of the above.

14) Downstream of the foreign protein expression gene cassette, at least one polyA addition selected from a polyA addition region of a fibroin H chain gene, a polyA addition region of a fibroin L chain gene, and a polyA addition region of a sericin gene The gene cassette for expression of a foreign protein according to any one of 1) to 13), wherein the gene cassette has a region.
15) An insect comprising a pair of piggyBac transposons with inverted repeats on both sides of the gene cassette for expressing a foreign protein according to any one of 1) to 14). Gene cassette for gene transfer into the chromosome of a cell.

16) An expression vector for insect cells, comprising the gene cassette for expressing a foreign protein according to any one of 1) to 14).
17) A gene transfer vector for insect cells, comprising the gene cassette for transferring genes into chromosomes of insect cells according to 15).
18) A method for producing a foreign protein, comprising introducing the gene transfer vector for insect cells according to 16) or 17) into insect cells.
19) The method for producing a foreign protein according to 18), wherein the insect cell is derived from a Lepidoptera insect.

20) The method for producing a foreign protein according to 19), wherein the insect cell is derived from Bombyx mori.
21) The method for producing a foreign protein according to 20), wherein the insect cells are silk gland cells of silkworm (Bombyx mori).
22) Utilizing the DNA transfer activity of piggyBac transposase, a recombinant silkworm in which the gene cassette for expression of the foreign protein was integrated into the chromosome was prepared, and the recombinant silkworm was used to introduce the recombinant silkworm into the silk gland or silk. 18. The method for producing a foreign protein according to 18), wherein after producing the protein, the foreign protein is recovered from the silk gland or the silk thread.

23) Simultaneous microinjection of a gene transfer vector for insect cells and DNA or RNA producing piggyBac transposase into silkworm eggs to produce a recombinant silkworm in which the gene cassette for expression of the foreign protein has been integrated into the chromosome 22. The method for producing a foreign protein according to 22) above.
24) A recombinant silkworm in which the gene cassette for expression of a foreign protein according to any one of the above 1) to 14) has been introduced into a chromosome and has the ability to produce a foreign protein in a silk gland or silk thread.

25) A silk thread containing a foreign protein produced by the recombinant silkworm described in 24) above.
26) The following polynucleotide (a) or (b):
(A) a polynucleotide consisting of the nucleotide sequence of position 1-4111 of SEQ ID NO: 2,
(B) a base sequence having one or several bases added, deleted or substituted with other bases in the base sequence (a), wherein the expression of a foreign gene is increased in silk gland tissue; Polynucleotide.

27) A polynucleotide characterized in that it hybridizes under stringent conditions with a polynucleotide consisting of the nucleotide sequence at positions 1-4111 of SEQ ID NO: 2, and that increases the expression of a foreign gene in silk gland tissue.
28) A method for increasing the expression of a foreign gene in silk gland tissue, comprising using the polynucleotide according to 26) or 27).

29) A gene cassette for expression of a foreign protein in which a promoter of a fibroin H chain gene is linked to the 3 ′ side of the polynucleotide according to the above 26) or 27) and a foreign protein structural gene is linked to the 3 ′ side thereof .
30) A gene cassette for introducing a gene into the chromosome of an insect cell, wherein a pair of piggyBac transposons is present on both sides of the gene cassette described in 29).
31) A recombinant silkworm in which the gene cassette according to 30) has been introduced into a chromosome, and which has the ability to produce a recombinant protein in a silk gland or silk thread.
32) A silk thread containing a foreign protein produced by the recombinant silkworm described in 31) above.
33) A silk thread containing a foreign protein (excluding GFP) produced by the recombinant silkworm according to 24) or 31).

  By introducing a gene cassette for expression in which the DNA sequence of the 5′-end portion and the DNA sequence of the 3′-end portion of the fibroin H chain gene are fused to a foreign protein gene into silk gland cells, etc., a large amount of foreign protein can be transferred into the silk gland cells. It has become possible to produce silk glands extracellularly and even silk. By this new method, a foreign protein production technology that can be easily purified was established by using a silk gland to produce a foreign protein without using a recombinant baculovirus.

  In the present invention, the “gene cassette for expression of a foreign protein” refers to a set of DNAs necessary for expression of a protein encoded by the foreign protein structural gene when introduced into an insect cell. This foreign protein expression cassette contains a foreign protein structural gene and a promoter that promotes the expression of the gene. Usually, it further contains a terminator and a poly-A additional region, preferably a promoter, a foreign protein structural gene, a terminator and a poly-A additional region. Further, a secretory signal gene linked to a foreign protein structural gene may be included between the promoter and the promoter. An arbitrary gene sequence may be linked to the poly-A additional sequence. It is also possible to link artificially designed and synthesized gene sequences.

  The term “gene cassette for gene transfer” refers to a gene cassette for expression of a foreign protein having an inverted repeat sequence of a pair of piggyBac transposons on both sides, and a piggyback (piggyBac) transposase. Means a set of DNAs introduced into insect cell chromosomes by the action of In the present invention, the expression vector for an insect cell includes a gene cassette for expressing a foreign protein or a gene cassette for introducing a gene, and a set of DNAs necessary for expressing the foreign protein when introduced into insect cells. Say. Also, in the present invention, the “insect cell gene transfer vector” includes a gene cassette for expression of a foreign protein or a gene cassette for gene transfer, and has a pair of piggyback transposon inverted repeat sequences at both ends, and And a DNA set introduced into the chromosome of insect cells by the action of piggyback transposase.

  The method for obtaining DNA used in the present invention is not particularly limited. A method of amplifying and obtaining necessary gene regions by using the PCR (polymerase chain reactiion) method based on known gene information, a method of screening based on known gene information from a genomic library or cDNA library using homology as an index, etc. Is mentioned. In the present invention, these genes also include genetic polymorphisms and mutants obtained by artificial mutation using a mutagen. Genetic polymorphism means that the nucleotide sequence of a gene is partially changed due to spontaneous mutation on the gene.

The promoter in the foreign protein expression cassette is not particularly limited, but a promoter having a high activity of promoting the expression of the foreign protein gene is preferable. For example, the promoter of the Drosophila heat shock protein gene and the promoter of the silkworm actin gene (Nature Biotechnology 18 , 81-84, 2000) described in JP-A-6-261770 and JP-A-62-285787 are exemplified. Fibroin H chain gene promoter (base numbers 255-574 of GenBank accession number V00094, base numbers 62118-62437 of GenBank accession number AF226688), fibroin L chain gene promoter (Gene, 100: 151-158: GenBank accession number) Promoters having high transcription promoting activity in silkworm silk gland cells, such as sericin gene promoter (base numbers 599 to 1656 of GenBank accession number AB007831), are preferred.

  In addition, high transcription promotion is observed by using the fibroin H chain gene promoter and its upstream region (base numbers 57444 to 62637 of GenBank accession number AF226688). It is well recognized that the region that promotes the transcriptional activity of such a gene retains its basic activity even when modified by various methods. Addition or deletion of one or more bases, artificially or spontaneously, to the extent that they have substantially the same function as the promoter that promotes the transcription activity of the gene and its upstream region, or DNA having a substitution with another base can also be used.

  “Addition or deletion of one or several bases or substitution with other bases” means that one or more bases are added or deleted, or one or more bases are compared with the base sequence. This means that the base has a mutation such as substitution of another base, but retains the same function as the base non-mutated sequence. Substantially equivalent functions in this case indicate high transcriptional activity and transcription promoting activity in silkworm silk gland cells. Specifically, in the rear silk gland or the middle silk gland of the silkworm on the 5th day and the second day, the transcription amount of the foreign protein gene in the recombinant silkworm having the mutated sequence is determined by the silkworm actin gene, the fibroin H chain gene or the fibroin L chain It shows that Northern analysis confirms that the transcription amount of either the gene or the sericin gene is within the range of 0.1 to 1 times that of either of them.

  Preferably, the amount is within the range of 0.1 to 1 times the transcription amount of the fibroin H chain gene. For comparison of the transcription amount, by using BIORAD's Molecular Imager FXPro, Amersham Pharmacia's Gene Images Random-Prime Labeling and Detection System can detect and compare the detected signal quantitatively. . At this time, the probe amount, hybridization conditions, and detection conditions are determined in advance so that the detection sensitivity of the foreign protein gene detection probe and that of the silkworm actin gene, fibroin H chain gene, fibroin L chain gene, or sericin gene detection probe are comparable. I do. Under these conditions, a probe for detecting a foreign protein gene and a probe for detecting a silkworm actin gene, a fibroin H chain gene, a fibroin L chain gene, or a sericin gene to be compared are hybridized, detected, and compared.

  Means for adding, deleting, or substituting one or several bases with other bases are known per se, for example, random mutagenesis, site-directed mutagenesis, homologous recombination, or polymerase. The chain amplification method (PCR) can be performed alone or in an appropriate combination. For example, a method of substituting cytosine base with uracil base by chemical treatment using sodium bisulfite, a method of performing PCR in a reaction solution containing manganese to reduce the accuracy of nucleotide incorporation during DNA synthesis, Various commercially available kits for specific mutagenesis can also be used.

  For example, Sambrook et al. [Molecular Cloning, Laboratory Manual 2nd Edition] Cold Spring Harbor Laboratory, 1989, Masami Muramatsu [Lab Manual Genetics] Maruzen Co., 1988, Ehrlich, HE. Principle and Application] The method can be carried out according to the method described in a book such as Stockton Press, 1989, or by modifying those methods.

  In addition, within the range having substantially the same function, a DNA that hybridizes under homologous sequences of these promoters and their upstream regions under stringent conditions can also be used. The single-stranded DNA hybridizes with the partner strand depending on the degree of homology. At this time, by setting stringency of the hybridization conditions more strictly, a sequence having higher homology can be specified. Stringent conditions are defined by the concentration of salt (eg, formamide) and the temperature in the hybridization and washing steps. The details are described in U.S. Patent No. 6,100,037. DNA identified by hybridization screening under such stringent conditions has a homology level of 70% or more, preferably 80% or more, and more preferably 90% or more.

  The “foreign protein structural gene” refers to a gene not possessed by the host cell in which the gene is to be expressed, and is a gene encoding a protein not originally produced by the host cell, and is not particularly limited. In view of industrial value, proteins produced by humans and mammals, such as genes for growth hormone, cytokines, growth factors, and cytoskeletal proteins, are mentioned. In addition, enzymes, genes of various proteins, and the like produced by microorganisms, plants, insects, and the like are also included in the scope of the present invention.

  In the gene cassette for expressing a foreign protein according to the present invention, the 5′-terminal portion of the fibroin H chain gene is a DNA sequence having an action of enhancing the expression of the foreign protein by the promoter, and the first exon and the first exon of the fibroin H chain gene are DNA sequence containing the entire intron or a part thereof and a part of the second exon. In the present invention, it has been clarified that the 5 'terminal portion of the fibroin H chain gene promotes gene expression and transcription in the posterior silk gland in a recombinant individual. It is well recognized that the region that promotes the transcriptional activity of such a gene retains its basic activity even when modified by various methods.

  Addition of one or more bases, whether artificial or spontaneous, to the 5'-terminal portion of the fibroin H chain gene that promotes the transcriptional activity of the gene, regardless of whether it has a substantially equivalent function, DNA having a deletion or substitution with another base can also be used. “Addition or deletion of one or several bases or substitution with other bases” means that one or more bases are added or deleted, or one or more bases are compared with the base sequence. This means that the base has a mutation such as substitution of another base, but retains the same function as the base non-mutated sequence.

  The substantially equivalent function in this case indicates a high transcription promoting activity in the silkworm silk gland cell. Means for adding, deleting, or substituting one or several bases with other bases are known per se, for example, random mutagenesis, site-directed mutagenesis, homologous recombination, or polymerase. The chain amplification method (PCR) can be performed alone or in an appropriate combination. For example, a method of substituting cytosine base with uracil base by chemical treatment using sodium bisulfite, a method of performing PCR in a reaction solution containing manganese to reduce the accuracy of nucleotide incorporation during DNA synthesis, Various commercially available kits for specific mutagenesis can also be used.

  For example, Sambrook et al. [Molecular cloning, Laboratory Manual 2nd Edition] Cold Spring Harbor Laboratory, 1989, Masamura Muramatsu [Lab Manual Genetics] Maruzen Co., 1988, Ehrlich, HE. [PCR Technology, Principle and Application of DNA Amplification] The method can be carried out according to the methods described in books such as Stockton Press, 1989, or by modifying those methods. Substantially equivalent functions in this case indicate high transcriptional activity and transcription promoting activity in silkworm silk gland cells.

  Specifically, in the rear silk gland or the middle silk gland of the silkworm on the 5th day and the second day, the transcription amount of the foreign protein gene in the recombinant silkworm having the mutated sequence is determined by the silkworm actin gene, the fibroin H chain gene, or the fibroin L chain. It shows that Northern analysis confirms that the transcription amount of either the gene or the sericin gene is within the range of 0.1 to 1 times that of either of them. Preferably, the amount is within the range of 0.1 to 1 times the transcription amount of the fibroin H chain gene.

  For comparison of the transcription amount, by using BIORAD's Molecular Imager FXPro, Amersham Pharmacia's Gene Images Random-Prime Labeling and Detection System can detect and compare the detected signal quantitatively. . At this time, the probe amount, hybridization conditions, and detection conditions are determined in advance so that the detection sensitivity of the foreign protein gene detection probe and that of the silkworm actin gene, fibroin H chain gene, fibroin L chain gene, or sericin gene detection probe are comparable. I do. Under these conditions, a probe for detecting a foreign protein gene and a probe for detecting a silkworm actin gene, a fibroin H chain gene, a fibroin L chain gene, or a sericin gene to be compared are hybridized, and detection and comparison are performed.

  Also, within a range having substantially the same function, a DNA in which a homologous sequence at the 5 'end of the fibroin H chain gene hybridizes under stringent conditions can be used. The single-stranded DNA hybridizes with the partner strand depending on the degree of homology. At this time, by setting stringency of the hybridization conditions more strictly, a sequence having higher homology can be specified. Stringent conditions are defined by the concentration of salt (eg, formamide) and the temperature in the hybridization and washing steps. The details are described in U.S. Patent No. 6,100,037. DNA identified by hybridization screening under such stringent conditions has a homology level of 70% or more, preferably 80% or more, and more preferably 90% or more.

  By fusing the 5 ′ side of the foreign protein structural gene to the 3 ′ side of this second exon so that the amino acid frame is continuous, the amount of foreign protein produced can be improved. If the length is too long, an extra amino acid residue is added to the N-terminal side of the target foreign protein, and the structure or activity of the target foreign protein may be lost. It is necessary to.

  In many cases, a favorable result can be obtained by setting the second exon portion immediately after the secretion signal gene of the fibroin H chain gene or up to several amino acid residues. Further, since it is considered that there is a region that enhances the promoter activity in the 5 ′ upstream region of the fibroin H chain promoter, that is, the 5.5 kbp upstream region, by adding this region, the expression of the foreign protein as the target protein can be achieved. An increase in volume can be expected.

  The gene structure of the silkworm fibroin H chain structural gene and the peripheral region has already been known (Nucleic acid research. 28, 2413-2419, 2000 / Genebank AF226688). In addition, analysis using a temporary transcription assay system using a cell extract revealed that it is approximately 238 bases upstream from the transcription start site (position 4971 in SEQ ID NO: 2) (described as -238; 4733 in SEQ ID NO: 2). (Proc. Natl. Acad. Sci. USA, 80, 7442-7446, 1983 / Proc. Natl. Acad. Sci. USA, 83, 9522-9526, 1986).

  However, up to now, a region further upstream than about 860 bases upstream from the transcription start point of the fibroin H chain gene (described as -860: position 4111 in SEQ ID NO: 2) efficiently promotes transcription in silk gland tissue. There is no example that clarified. In the present invention, the region further upstream than about 860 bases upstream (-860) from the transcription start point of the fibroin H chain gene is examined and analyzed, and the region further upstream than about 860 bases upstream from the transcription start point of the fibroin H chain gene is It has been revealed that transcription in silk gland tissue is efficiently promoted, and that the amount of foreign recombinant protein produced in silk gland tissue is improved, and that the amount of foreign recombinant protein produced in silk is increased. It was also found to improve Specifically, the nucleotide sequence from position 1-4111 in SEQ ID NO: 2 efficiently promotes transcription in the silk gland tissue and improves the production of the foreign recombinant protein in the silk gland tissue. Further, it has been found that the amount of production of a foreign recombinant protein in a silk thread is also improved.

  By adding this region, an increase in the expression level of the foreign protein as the target protein can be expected. In the 5 'upstream region of the fibroin H chain promoter, that is, the 5.5 kbp upstream region, a polynucleotide comprising the nucleotide sequence at position 1-44674 of SEQ ID NO: 2 (base number 57444 of AF226688) is preferably used as the nucleotide sequence for enhancing the promoter activity. 6262117). More preferably, a polynucleotide consisting of the base sequence at positions 1-4111 of SEQ ID NO: 2 (corresponding to base numbers 57444 to 61554 of AF226688) is used.

  As long as the polynucleotide consisting of the nucleotide sequence of positions 1-4111 of SEQ ID NO: 2 has a substantially equivalent function, addition or deletion of one or more bases, whether artificial or spontaneous, It is also possible to use DNA that has been lost or has been substituted with another base. “Addition or deletion of one or several bases or substitution with other bases” means that one or more bases are added or deleted, or one or more bases are compared with the base sequence. This means that the base has a mutation such as substitution of another base, but retains the same function as the base non-mutated sequence.

  In this case, the substantially equivalent function means that the polynucleotide comprising the nucleotide sequence at positions 1-4111 of SEQ ID NO: 2 has an effect of increasing the expression of a foreign gene in a silk gland tissue or a posterior silk gland tissue. Represent. The effect of increasing the expression of the foreign gene in the silk gland tissue or posterior silk gland tissue is observed as an effect of increasing the amount of protein derived from the linked gene by promoting transcriptional activity. Specifically, as compared with the case where no polynucleotide is used, the effect of increasing the production amount of the protein derived from the ligated gene in the rear silk gland of the fourth day of the fifth stage of silkworm by at least two times or more is shown.

  For comparison of the amount of protein production, by using a molecular imager FXPro manufactured by BIORAD, it is possible to detect and compare signals derived from a foreign protein detected by an ECL Western Blotting Detection System manufactured by Amersham Pharmacia.

  Means for adding, deleting, or substituting one or several bases with other bases are known per se, for example, random mutagenesis, site-directed mutagenesis, homologous recombination, or polymerase. The chain amplification method (PCR) can be performed alone or in an appropriate combination. For example, a method of substituting cytosine base with uracil base by chemical treatment using sodium bisulfite, a method of performing PCR in a reaction solution containing manganese to reduce the accuracy of nucleotide incorporation during DNA synthesis, Various commercially available kits for specific mutagenesis can also be used.

  For example, Sambrook et al. [Molecular Cloning, Laboratory Manual Second Edition] Cold Spring Harbor Laboratory, 1989, Masamura Muramatsu [Lab Manual Genetic Engineering] Maruzen Co., 1988, Ehrlich, HE. [PCR Technology, Principle and Application of DNA Amplification] The method can be carried out according to the methods described in books such as Stockton Press, 1989, or by modifying those methods.

  In addition, a DNA to which a polynucleotide consisting of the nucleotide sequence of positions 1-4111 of SEQ ID NO: 2 hybridizes under stringent conditions can also be used within a range having substantially the same function. The single-stranded DNA hybridizes with the partner strand depending on the degree of homology. At this time, by setting stringency of the hybridization conditions more strictly, a sequence having higher homology can be specified. Stringent conditions are defined by the concentration of salt (eg, formamide) and the temperature in the hybridization and washing steps. The details are described in U.S. Patent No. 6,100,037. DNA identified by hybridization screening under such stringent conditions has a homology level of 70% or more, preferably 80% or more, and more preferably 90% or more.

  The 3'-terminal portion of the fibroin H chain gene is a DNA sequence having an effect of secreting a large amount of the foreign protein out of the silk gland cells when the foreign protein is produced in the silk gland. The recombinant silkworm, in which a gene cassette for expression of a foreign protein obtained by fusing the 3 ′ end of the fibroin H chain gene, which is a secretion signal to the silk to the 3 ′ side, is introduced into the chromosome, produces the foreign protein in the silk. It is possible to do. Further, the 3′-terminal portion of the fibroin H chain gene may be located upstream, downstream, or in the foreign protein gene of the foreign protein gene.

  At least one cysteine residue is present in this portion, and when the 3 ′ end of the fibroin H chain gene is used as it is, the cysteine residue is located at the 20th position from the carboxyl end of the fibroin H chain protein. Become. This cysteine has a role of binding to the fibroin L chain via a disulfide bond. The length of the DNA sequence at the 3 ′ end of the fibroin H chain gene is not particularly limited as long as it does not inhibit the formation of disulfide bonds with the fibroin L chain. Since the fibroin H chain has a repeat sequence of DNA at least about 100 bases upstream from the 3 ′ end, it is difficult to cut or process the DNA sequence at this upstream portion to an arbitrary length with a restriction enzyme. It is.

  Therefore, from the viewpoint of easiness of the genetic engineering technique, about 100 base pairs on the 3 ′ side where the repetitive DNA sequence of the fibroin H chain gene has been completed can be suitably used as the 3 ′ end of the fibroin H chain. In addition, if the 3′-terminal portion of the fibroin H chain gene is long, many amino acids derived from the carboxyl terminal of the fibroin H chain protein will bind to the carboxyl terminal of the foreign protein, and the structure and activity of the target foreign protein will be lost. There are cases. Therefore, depending on the target protein, the DNA sequence of the 3 ′ terminal of the fibroin H chain gene may need to be as short as possible.

  In the present invention, it has been revealed that the 3′-terminal portion of the fibroin H chain is a region encoding a protein having an effect of secreting a foreign protein into silk. Addition or deletion of one or more bases, or addition or deletion of other bases, whether artificial or spontaneous, to the extent that the fibroin H chain 3 ′ end has substantially the same function. DNA having a substitution can also be used.

  “Addition or deletion of one or several bases or substitution with other bases” means that one or more bases are added or deleted, or one or more bases are compared with the base sequence. This means that the base has a mutation such as substitution of another base, but retains the same function as the base non-mutated sequence. The substantially equivalent function in this case indicates that the protein region formed from the 3'-terminal portion of the fibroin H chain has an effect of causing the silk protein to secrete a foreign protein.

  The measurement of the effect of exogenous protein secretion into silk is measured by using BIORAD's Molecular Imager FXPro to detect and compare the signal derived from the exogenous protein detected by Amersham Pharmacia's ECL Western Blotting Detection System. it can.

  Means for adding, deleting, or substituting one or several bases with other bases are known per se, for example, random mutagenesis, site-directed mutagenesis, homologous recombination, or polymerase. The chain amplification method (PCR) can be performed alone or in an appropriate combination. For example, a method of substituting cytosine base with uracil base by chemical treatment using sodium bisulfite, a method of performing PCR in a reaction solution containing manganese to reduce the accuracy of nucleotide incorporation during DNA synthesis, Various commercially available kits for specific mutagenesis can also be used.

  For example, edited by Sambrook et al. [Molecular Cloning, Laboratory Manual 2nd Edition] Cold Spring Harbor Laboratory, 1989, Masanori Muramatsu [Lab Manual Genetic Engineering] Maruzen Co., 1988, Ehrlich, HE. [PCR Technology, Principle and Application of DNA Amplification] The method can be carried out according to the methods described in books such as Stockton Press, 1989, or by modifying those methods.

  In addition, a DNA that can hybridize under stringent conditions with a homologous sequence of the 3′-terminal portion of the fibroin H chain can also be used as long as it has substantially the same function. The single-stranded DNA hybridizes with the partner strand depending on the degree of homology. At this time, by setting stringency of the hybridization conditions more strictly, a sequence having higher homology can be specified. Stringent conditions are defined by the concentration of salt (eg, formamide) and the temperature in the hybridization and washing steps. The details are described in U.S. Patent No. 6,100,037. DNA identified by hybridization screening under such stringent conditions has a homology level of 70% or more, preferably 80% or more, and more preferably 90% or more.

There is no particular limitation on the poly A region, but a poly A region of a protein gene such as fibroin H chain, fibroin L chain, and sericin, which is expressed in a large amount in a silk gland, can be preferably used.
The vector in the present invention refers to a vector having a circular DNA structure or a linear DNA structure. In particular, a vector that can replicate in Escherichia coli and has a circular DNA structure can be suitably used. For the purpose of facilitating the selection of transformants, a marker gene such as an antibiotic resistance gene or a jellyfish-derived fluorescent green protein gene can be incorporated into this vector.

  The insect cells used in the present invention are not particularly limited, but are preferably lepidopteran insects, more preferably silkworm (Bombyx mori) -derived cells, still more preferably silkworm silk gland cells or silkworm (Bombyx mori) eggs. The cells that are included. Among the silk gland cells, the posterior silk gland cells of silkworm fifth-instar larvae, in which the synthesis of fibroin protein is active and which are easy to handle, are preferred.

  The method for introducing the gene cassette and vector for expression of a foreign protein into insect cells is not particularly limited. As a method for introducing into insect cultured cells, a calcium phosphate method, a method using electroporation, a method using a liposome, a method using a gene gun, a method using microinjection, and the like can be used. For example, a gene can be easily introduced into a posterior silk gland tissue taken out from the body of a fifth-instar silkworm larva by using a gene gun.

Gene transfer into the posterior silk gland by using a gene gun can be performed, for example, by using a gold particle coated with a vector containing a gene cassette for expression of a foreign protein using a particle gun (model number: PDS-1000 / He) manufactured by Bio-Rad. This can be achieved by injecting the posterior silk gland fixed on an agar plate or the like at a He gas pressure of 1,100 to 1,800 psi.
When a gene is introduced into cells contained in the silkworm moth (Bombyx mori) egg, a microinjection method is suitable. Here, when microinjection is performed on an egg, it is not necessary to directly perform microinjection on cells in the egg, and the gene can be introduced only by microinjection into the egg.

By microinjecting the vector having the `` gene cassette for gene transfer '' of the present invention into eggs of silkworm (Bombyx mori), a recombinant silkworm in which the `` gene cassette for expression of a foreign protein '' of the present invention has been introduced into a chromosome is obtained. It is possible to get. For example, according to the method of Tamura et al. (Nature Biotechnology 18 , 81-84, 2000), a vector having a “gene cassette for gene transfer” and a plasmid in which a piggyBac transposase gene is placed under the control of the silkworm actin promoter are used. At the same time, the eggs of the silkworm are microinjected, the hatched larvae are bred, and the obtained adults (G0) are crossed within the group to obtain the next generation (G1) silkworm larvae.

  Recombinant silkworms usually appear at a frequency of 1-2% in this G1 generation. Recombinant silkworms can be selected by extracting DNA from a part of the tissue of the G1 generation silkworm and performing PCR using primers designed based on the foreign protein gene. Alternatively, if a gene encoding a green fluorescent protein (eg, GFP and its analogs) linked downstream of a promoter that can be expressed in silkworm cells has been introduced into the “gene transfer gene cassette” in advance, the G1 generation Selection of recombinant silkworms can be easily performed by selecting individuals that emit green fluorescence under ultraviolet light for silkworms, for example, the first instar larvae.

Examples of a promoter capable of expressing a gene encoding a green fluorescent protein, which can be expressed in silkworm cells, include a promoter of a Drosophila heat shock protein gene and a silkworm actin gene described in JP-A-6-261770 and JP-A-62-285787. (Nature Biotechnology 18 , 81-84, 2000), and 3xP3 promoter known to be expressed in the Drosophila optic nerve. Preferably, the promoter of the silkworm actin gene (Nature Biotechnology 18 , 81-84, 2000) or the 3xP3 promoter is used.

  In addition, as a promoter that can be expressed in silkworm cells, a promoter having high transcription promoting activity in silkworm silk gland cells, for example, a promoter of a fibroin H chain gene (base numbers 255 to 574 of GenBank accession number V00094, GenBank accession number AF226688) Selection of the promoter of the fibroin L chain gene (Gene, 100: 151-158: GenBank accession number M76430) and the promoter of the sericin gene (Base numbers 599 to 1656 of GenBank accession number AB007831) Is also possible.

  However, according to the present invention, when a promoter having a high transcription promoting activity in a silkworm silk gland cell is linked to a gene encoding a green fluorescent protein, a large amount of green fluorescent protein is produced in the silkworm silk gland cell. In addition, a green fluorescent protein is produced in the silk thread in addition to the foreign protein of interest. In this case, when purifying and recovering the target foreign protein from the silk thread and the silk thread, the green fluorescent protein makes purification and recovery of the target foreign protein difficult as a foreign protein. Further, it is conceivable that the green fluorescent protein may impart undesired properties to the physical properties of the silk thread, such as a decrease in elongation.

  In addition, in order to obtain a recombinant silkworm in which the `` gene cassette for expression of a foreign protein '' has been introduced into the chromosome, in microinjecting a vector having the `` gene cassette for gene transfer '' into the silkworm (Bombyx mori) egg, Simultaneous microinjection of PiggyBac transposase proteins can also obtain recombinant silkworms in the same manner as described above.

  The PiggyBac transposon is a transposable element of DNA having an inverted sequence of 13 base pairs at both ends and an ORF of about 2.1 k base pairs inside. The piggyback (PiggyBac) transposon used in the present invention is not particularly limited. For example, those derived from Trichoplusia ni cell line TN-368, Autographa californica NPV (AcNPV), and Galleria mellonea NPV (GmMNPV) can be used. Preferably, a plasmid pHA3PIG having a part of a piggyback (PiggyBac) derived from Trichoplusia ni cell line TN-368 and pPIGA3GFP (Nature biotechnology 18, 81-84, 2000) are used, and the piggyback transposase having its gene and DNA transfer activity is used. Can be prepared.

  The genetically modified silkworm used in the present invention is a silkworm in which a foreign protein gene has been introduced into a silkworm chromosome, and after treating the silkworm chromosomal DNA with a restriction enzyme according to a conventional method, the foreign protein gene labeled according to a conventional method Is a silkworm that gives a positive signal when Southern blotting is performed using as a probe. The gene locus on the chromosome into which the foreign protein gene is introduced is not particularly limited as long as it does not inhibit the development, differentiation, and growth of the silkworm. The recombinant silkworm has the ability to produce foreign proteins in its silk gland cells, silk gland lumen, and silk. In addition, the recombinant silkworm can develop normally, can be crossed, stably retain the introduced foreign protein gene, and transmit it to progeny.

  Therefore, it is possible to easily scale up the amount of foreign protein produced by increasing the number of transgenic silkworms. In mating, it is also possible to improve the amount of foreign protein produced by mating with a wild-type silkworm. In this case, it is necessary to pass the silkworms into which the desired foreign protein gene has been introduced, while appropriately selecting them. In this case, the presence or structure of the marker gene or foreign protein gene used for the selection of recombinant silkworms can be easily analyzed by PCR, Southern blotting, etc., using the DNA of cells obtained from any tissue. It is possible to determine the offspring that inherited the transgenic silkworm gene.

  Insect cells and silkworm silk glands into which the gene cassette for expression of a foreign protein of the present invention has been introduced can be produced in the culture supernatant or cells by culturing each in a culture solution suitable for culture. It is possible. For example, BmN cells, which are silkworm ovary-derived cells into which the gene cassette for expression of the present invention has been introduced, are cultured at 27 ° C. in a TC-100 medium (manufactured by Pharmingen), and cultured for 3 to 4 days. To produce the desired foreign protein. Further, the posterior silk gland of the silkworm, for example, is aseptically extracted from the fifth-instar larvae and then cultured at 25 ° C. in an Insect Grace's medium to produce a foreign protein. When performing protein production in the silk gland, it is preferable to maintain the dissolved oxygen concentration in the medium at a high level.Factors that inhibit the synthesis of low molecular weight proteins that accumulate in the medium are, for example, ultrafiltration membranes. It is preferable to culture the cells while removing them, for example, so that the protein can be synthesized for a long time.

The silk gland into which the foreign protein gene into which the 3′-terminal portion of the fibroin H chain gene of the present invention is fused can introduce large amounts of the desired foreign protein into the culture supernatant. Since the contaminating protein in the silk gland culture supernatant is almost only fibroin, it is easy to purify the target protein from the silk gland culture supernatant, and as a result, a highly pure target protein can be obtained. .
The recombinant silkworm obtained in the present invention can be bred in the same manner as a normal silkworm, and it is possible to produce a foreign protein by rearing it under normal conditions. Depending on the target foreign protein, it is also possible to improve the production amount of the foreign protein by optimizing the culture temperature, humidity, feeding conditions, etc., especially at the 5th stage.

  The recombinant silkworm, into which the foreign protein gene obtained by fusing the 3′-terminal portion of the fibroin H chain gene of the present invention is introduced, can produce a large amount of the desired foreign protein in its cocoon. From the obtained cocoon, the target foreign protein can be easily purified and recovered. In addition, depending on the function of the produced foreign protein, the obtained silk thread containing the foreign protein can be used in various industrial applications as it is or in a partially processed form.

  A foreign protein can be obtained from the silk gland or cocoon of the recombinant silkworm obtained by the present invention by an appropriate extraction operation. There is no particular limitation on the solvent used to extract the foreign protein from the silk gland or cocoon thread, but in many cases, an aqueous solvent system is preferred. The aqueous solution used for extraction can contain appropriate solutes to facilitate the extraction of foreign proteins. For example, inorganic acids such as phosphoric acid, organic acids such as acetic acid, citric acid, and malic acid; salts such as salt, urea, guanidine hydrochloride, and calcium chloride; and polar organic solvents such as ethanol, methanol, acetonitrile, and acetone. Can be The pH of the extraction solution is not particularly limited, and any pH can be used as long as it does not deactivate the function of the target foreign protein.

  The method for isolating and purifying the extracted foreign protein is not particularly limited, and a normal protein purification method can be used. For example, while using the intrinsic function of the intended useful protein as an index, silica gel carriers, ion-exchange carriers, gel filtration carriers, chelating carriers, chromatography using a dye-supporting carrier and the like, ultrafiltration, gel filtration, Purification and isolation can be achieved by a combination of dialysis, desalting by salting out, and concentration. For example, feline interferon-ω can be collected from a silkworm gland or cocoon of a silkworm into which feline interferon-ω gene has been introduced into a potential fraction obtained by homogenizing a 20 mM phosphate buffer (pH 7.0). it can. Furthermore, the purity of feline interferon-ω can be increased by adsorbing the obtained extract to, for example, a blue sepharose carrier, washing, and eluting with a buffer containing salts.

  The cytokines produced in this way can be used for pharmaceutical applications, various measurements and diagnostic applications, similarly to cytokines produced by other conventional production methods. In this case, you may use as a mixture which added various additives. Silkworm tissues or cocoons expressing cytokines can be used as they are or processed, and used as medical or clothing fibers. In addition, the tissue or silk thread of the recombinant silkworm expressing the enzymes can be used for the enzyme reaction as it is.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
Example 1. Preparation of Bombyx mori genomic DNA
The silkworm, 5 days old and 3 days, was dissected and the posterior silk gland tissue was removed. After washing with 1 × SSC, 200 μl of a DNA extraction buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 100 mM NaCl) was added. Proteinase K (final 200 μg / ml) was added, the tissue was thoroughly ground with a grinder, 350 μl of DNA extraction buffer and 60 μl of 10% SDS were added, mixed, and incubated at 50 ° C. for 2 hours. After 500 μl of Tris-HCl saturated phenol pH 8.0 was added and mixed for 10 minutes, the mixture was centrifuged at 10,000 rpm for 5 minutes at 4 ° C., and the supernatant was recovered.

  An equal amount of phenol / chloroform / isoamyl alcohol (25: 24: 1) was added to the supernatant, mixed, and centrifuged. Phenol / chloroform / isoamyl alcohol was added again, and the supernatant was collected after centrifugation. After adding and mixing an equal amount of chloroform / isoamyl alcohol (24: 1), chloroform / isoamyl alcohol was added again to the centrifuged supernatant, and the supernatant was collected after centrifugation. 1/10 volume of 3M sodium acetate (pH 5.2) was added and mixed to the obtained supernatant, and further 2.5 volumes of cold ethanol was added. And genomic DNA was precipitated. After washing the DNA precipitate with 70% ethanol, it was air-dried. Genomic DNA solution was prepared by dissolving and diluting with RNase-containing sterilized water to 100 μg / ml.

Example 2 Preparation of Gene The used gene was obtained by preparing primers of both ends using known sequences and performing PCR using an appropriate DNA source as a template. A restriction enzyme cleavage site was added to the end of the primer for subsequent genetic manipulation.
The fibroin H chain promoter (base numbers 62118 to 62637 of GeneBank accession number AF226688: the following P region) is composed of two types of primers 4 (SEQ ID NO: 4) and 5 (SEQ ID NO: 5) using Bombyx mori genomic DNA as a template. It was obtained by PCR using primers.

Fibroin H chain promoter, fibroin H chain gene first exon, first intron, second exon region (base numbers 62118 to 63513 of GeneBank accession number AF226688: hereinafter HP region) are obtained by using Bombyx mori genomic DNA as a template and primer 4 (SEQ ID NO: 4) and primer 10 (SEQ ID NO: 10) were obtained by PCR using two types of primers.
The fibroin H chain upstream promoter, the first exon and the first intron region of the fibroin H chain gene (base numbers 57444 to 62927 of GeneBank accession number AF226688: hereinafter referred to as the HUP region) are obtained by using Bombyx mori genomic DNA as a template and primer 12 (SEQ ID NO: 12) and a primer 13 (SEQ ID NO: 13).

  The feline interferon-ω gene (base numbers 9 to 593 of GeneBank accession number S62636: the following IC region) is composed of primer 6 (SEQ ID NO: 6) and primer 7 (SEQ ID NO: 6) using baculovirus rBNV100 encoding feline interferon-ω gene as a template. It was obtained by PCR using the two types of primers in 7). rBNV100 can be obtained by excising the gene for feline interferon-ω from a plasmid extracted from, for example, E. Coli (pFeIFN1) (Jiko No. 1633) and using a silkworm cloning vector (T. Horiuchi et al., Agric. Biol. Chem. , 51, 1573-1580, 1987), and the recombinant plasmid and the silkworm polynucleotic disease virus DNA can be co-transfected into silkworm established cells.

The fibroin H chain poly A signal region (base numbers 79201 to 79995 of GeneBank accession number AF226688: hereinafter referred to as A region) is composed of primers 8 (SEQ ID NO: 8) and 9 (SEQ ID NO: 9) using Bombyx mori genomic DNA as a template. It was obtained by PCR using two types of primers.
The fibroin H chain C-terminal region gene / fibroin H chain poly A signal region (base numbers 79099 to 79995 of GeneBank accession number AF226688: hereinafter HA region) is composed of Bombyx mori genomic DNA as a template and primer 11 (SEQ ID NO: 11). It was obtained by PCR using two types of primers, primer 9 (SEQ ID NO: 9).

The β-galactosidase (β-gal) gene was obtained by PCR using two types of primers, primer 16 (SEQ ID NO: 16) and primer 17 (SEQ ID NO: 17), using pβgal-Basic vector (Clontech) as a template. .
PCR was performed using KODplus (manufactured by Toyobo Co., Ltd.) according to the attached protocol. That is, each template, 100 ng for Bombyx mori genomic DNA, 10 ng for Bombyx mori posterior silk gland cDNA and pβgal-Basic vector, 50 pmol of each primer, 10 μl of attached 10 × PCR buffer, Each reagent is added to 1 mM MgCl 2, 0.2 mM dNTPs, and 2 units KODplus to make a total volume of 100 μl. 30 cycles of DNA denaturation at 94 ° C for 15 seconds, primer annealing at 55 ° C for 30 seconds, and extension at 68 ° C for 60 to 300 seconds using a Perkin-Elmer DNA thermal cycler. I let it.

  These reaction solutions were electrophoresed on a 1% agarose gel, and about 0.3 kbp in the P region, about 1.4 kbp in the HP region, about 5.5 kbp in the HUP region, about 580 bp in the IC region, and about 580 bp in the A region, respectively. A DNA fragment of 0.8 bp, about 0.9 bp in the HA region, and about 3.2 kbp in the β-gal gene was extracted and prepared according to a conventional method. These DNA fragments were phosphorylated by polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.), digested with HincII, and then dephosphorylated. The pUC19 vector was treated at 16 ° C. using DNA Ligation Kit Ver.2 of Takara Shuzo Co., Ltd. The reaction was performed overnight and ligated. Using these, Escherichia coli was transformed according to a conventional method, and it was confirmed that the PCR fragment was inserted into the obtained transformant by performing PCR on the obtained colonies under the same conditions as described above. The inserted plasmid was prepared by a conventional method. By sequencing these plasmids, it was confirmed that the obtained fragments had the nucleotide sequences of the respective genes.

Example 3 Preparation of Plasmid for Expression of β-Galactosidase The plasmid having the β-gal gene prepared in Example 2 was cut with Sal I and Hind III, and the plasmid having a fibroin H chain promoter was replaced with Sal I and Hind III. A fragment (P region) of about 0.3 kbp. Further, this was cut with BamHI, and a fragment (A region) of about 0.8 kbp cut out from the plasmid having a fibroin H chain polyA signal region was cut out with BamHI, and the resulting plasmid having a β-gal gene was inserted. Was purified using a QIAGEN Plasmid Maxi Kit according to the attached protocol. The resulting plasmid was named pPgalA, and was confirmed to be the target plasmid by PCR and sequencing.

  Similarly, the plasmid having the β-gal gene prepared in Example 2 was cleaved with Sal I and Hind III, which had the fibroin H chain promoter, the fibroin H chain gene first exon, first intron, and second exon region. An approximately 1.4 kbp. Fragment (HP region) cut out from the plasmid with Sal I and Hind III was inserted. This was further cut with BamH I, and a fragment (HA region) obtained by inserting about 0.9 kbp.fragment (HA region) cut out with BamH I from a plasmid having a fibroin H chain C-terminal region and a fibroin H chain polyA signal region was obtained. The plasmid having the β-gal gene was purified using the QIAGEN Plasmid Maxi Kit according to the attached protocol. The resulting plasmid was named pHPgalHA, and was confirmed to be the target plasmid by PCR and sequencing.

Example 4. Preparation of plasmid for gene introduction PigA3GFP (Nature Biotechnology 18 , 81-84, 2000) was used for the plasmid for gene introduction. That is, the region encoding transposase was removed from the plasmid p3E1.2 disclosed in US Pat. No. 6,218,185, and the A3 promoter (base numbers 1764 to 2595 of GenBank accession number U49854) and the pEGFP-N1 vector (Clontech) PigA3GFP into which a GFP-derived SV40-derived polyA additional sequence (nucleotide numbers 659 to 2578 of GenBank accession number U55762) is inserted, and this vector can be distributed from the National Institute of Agrobiological Resources. is there. The XhoI site on the upstream side of the A3 promoter was blunted, and the feline interferon-ω gene expression cassette was inserted.

  The configuration of the gene expression cassette in this example is the fibroin H chain promoter, feline interferon-ω, fibroin H chain polyA signal region (P, IC, A), or the fibroin H chain promoter, fibroin H chain gene first exon, First intron, second exon region, feline interferon-ω, fibroin H chain C-terminal region, fibroin H chain poly A signal region (HP, IC, HA), or fibroin H chain upstream promoter, fibroin H chain gene first exon・ First intron ・ Second exon region ・ Feline interferon-ω ・ Fibroin H chain C terminal region ・ Fibroin H chain polyA signal region (HUP ・ IC ・ HA) or fibroin H chain promoter ・ Fibroin H chain gene first exon ・1st intron, 2nd exon area, cat interface Heron-ω / fibroin H chain polyA signal region (HP ・ IC ・ A).

The specific method will be described below.
The construction of the P-IC-A construct was performed by the following method. The plasmid having the feline interferon-ω (IC region) prepared in Example 2 was digested with Sal I and Hind III, and the plasmid having the fibroin H chain promoter was cut out with Sal I and Hind III to obtain a fragment of about 0.3 kbp. (P region) was inserted. This was further cut with BamH I, and an approximately 0.8 kbp. Fragment (A region) cut out with BamH I from a plasmid having a fibroin H chain polyA signal region was inserted therein. This plasmid having PIC.A was cleaved with AscI, and the excised approximately 1.7 kbp fragment was blunted with T4 DNA Polymerase from Takara Shuzo Co., Ltd., cleaved with XhoI, blunted, and dephosphorylated. PigA3GFP was ligated to construct a gene introduction construct containing a PICA gene cassette. The method is shown in FIG. 1 and FIG.

  The HP / IC / HA construct was prepared by the following method. The plasmid having feline interferon-ω (IC region) prepared in Example 2 was digested with Sal I and Hind III, and the fibroin H chain promoter / fibroin H chain gene first exon, first intron, second exon region Approximately 1.4 kbp. Fragment (HP region) cut out from plasmid having SalI and HindIII was inserted.

  This was further cut with BamH I, and an approximately 0.9 kbp fragment (HA region) cut out with BamH I from a plasmid having a fibroin H chain C-terminal region and a fibroin H chain polyA signal region was inserted therein. The plasmid having HP, IC, and HA was digested with Asc I, and the excised 2.9 kbp fragment was blunted with T4 DNA Polymerase from Takara Shuzo Co., Ltd., and digested with Xho I, and then blunted and dephosphorylated. PigA3GFP was ligated to construct a gene introduction construct containing the HP / IC / HA gene cassette. The method is shown in FIG. 3 and FIG.

  The preparation of the HUP / IC / HA construct was performed by the following method. Using 1 ng of the HP / IC / HA construct as a template, fibroin H chain first intron, second exon region, and feline interferon were obtained by PCR using primer 14 (SEQ ID NO: 14) and primer 15 (SEQ ID NO: 15). -Ω, fibroin H chain C-terminal region, fibroin H chain poly A signal region, about 2.1 kbp.

  This was cleaved with Xho I and Sph I, and a fragment containing about 5.5 kbp fragment (HUP region) cut out with Xho I and Sph I from a plasmid having a fibroin H chain upstream promoter, a fibroin H chain gene first exon and a first intron. ) Was inserted. The plasmid having the HUP / IC / HA was digested with Asc I, and the excised approximately 7.6 kbp fragment was blunted with T4 DNA Polymerase from Takara Shuzo Co., Ltd., and then digested with Xho I, and then blunted and dephosphorylated. PigA3GFP was ligated to construct a gene transfer construct containing the HUP / IC / HA gene cassette. The method is shown in FIGS.

  The HP / IC / A construct was produced by the following method. The plasmid having feline interferon-ω (IC region) prepared in Example 2 was digested with Sal I and Hind III, and the fibroin H chain promoter / fibroin H chain gene first exon, first intron, second exon region Approximately 1.4 kbp. Fragment (HP region) cut out from plasmid having SalI and HindIII was inserted.

  This was further cut with BamH I, and an approximately 0.8 kbp. Fragment (A region) cut out with BamH I from a plasmid having a fibroin H chain polyA signal region was inserted therein. The plasmid having HP, IC and A was digested with Asc I, and the excised approximately 2.8 kbp fragment was blunted with T4 DNA Polymerase from Takara Shuzo Co., Ltd. and digested with Xho I, followed by blunting and dephosphorylation. PigA3GFP was ligated to construct a gene introduction construct containing the HP / IC / A gene cassette. The method is shown in FIG. 7 and FIG.

  Using the QIAGEN Plasmid Maxi Kit, attach the P / IC / A gene transfer construct, HP / IC / HA gene transfer construct, HUP / IC / HA gene transfer construct, and HP / IC / A gene transfer construct Purified according to

Example 5. Expression of β-galactosidase in silkworm silk gland Gold particles having a diameter of 1.6 μm were washed with 100% ethanol, sterilized, and suspended in sterilized distilled water (60 mg / ml). Introduction of the β-gal gene expression cassette into the silkworm silk gland was performed using a gene gun. That is, 50 ul (0.3 mg) of gold particles, 10 ug of the expression plasmid pPgalA or pHPgalHA obtained in Example 3, 50 ul of 2.5 M calcium chloride, and 20 ul of 0.1 M spermidine were sequentially mixed and left at room temperature for 30 minutes. The gold particles coated with pHgalC were collected by centrifugation.

  The obtained gold particles were washed twice with 70% ethanol and then dispersed in 50 ul of 100% ethanol. 10 ul of the gold particle suspension was placed on the microcarrier and dried. As a gene gun, PDS-1000 / He manufactured by BIO-RAD was used. The posterior silk gland excised from the silkworm larvae on the 3rd day of the 5th day was washed twice with PBS, placed on a 1% agar plate, and sprayed with gold particles coated with DNA at a pressure of 1,100 psi. After DNA introduction, the silk gland was transferred to a 20 ml Grace Insect medium and cultured at 25 ° C. for 2 days. After the culture, the culture supernatant and the silk gland cells were collected, and the expression of β-gal was confirmed.

  Expression was confirmed by Western analysis. Silk gland cells were homogenized in PBS to extract the cell contents. The culture supernatant and the cell extract were both adjusted to a total protein concentration of 1.0 mg / ml, and SDS-PAGE was performed using these as samples. After blotting the membrane, β-gal protein was detected using ECL Plus ™ Western blotting Kit (Amersham Pharmacia) according to the attached protocol. That is, the blotted membrane was blocked overnight at 4 ° C. in a blocking solution (5% skim milk / 0.1% Tween 20 / PBS).

  The membrane was washed twice with TPBS (0.1% Tween 20 / PBS) and treated with an anti-β-gal protein antibody (manufactured by Sigma) diluted 1000-fold with TPBS for 1 hour at room temperature. The membrane was washed twice with TPBS and further three times for 5 minutes with TPBS. After that, the mixture was diluted 10,000 times with TPBS, and then treated with an HRP-labeled anti-rabbit IgG antibody for 1 hour at room temperature. After washing the membrane twice with TPBS and further three times for 5 minutes with TPBS, a detection reagent (solution A + solution B) of ECL Plus ™ Western blotting Detection System (manufactured by Amersham Pharmacia) was added. The luminescence was exposed to HyperfilmTM ECLTM and developed. For signal comparison, a molecular imager FXPro manufactured by BIORAD was used.

  Since β-gal protein was detected only in the silk gland cells into which pHPgalHA was introduced and in the culture supernatant, the region other than the fibroin H chain promoter, ie, the first exon of the fibroin H chain gene, was used for protein synthesis or gene expression in cells. -It became clear that the first intron and the second exon region play important roles. Extracellular secretion was also confirmed. The result is shown in FIG.

Example 6 Preparation of Genetically Modified Silkworm 0.5 mM phosphate buffer (pH 7.0) containing 200 μg / ml of each of the plasmid for gene transfer described in Example 4 and DNA (pHA3PIG) for producing piggyback transposase protein. A 5 mM KCl solution was prepared, and 3 to 20 nl was microinjected into 500 silkworm eggs within 4 hours after laying eggs. pHA3PIG (Nature biotechnology 18,81-84,2000 available from National Institute for Agrobiological Resources) lacks one inverted repeat sequence of the piggyback transposon, the 5 'flanking region, and the leader sequence of the transposase gene. Instead, the 5 'flanking region and leader sequence of the silkworm actin gene have been integrated. pHA3PIG has the function of producing a piggyback transposase protein by the action of the actin promoter, but does not transfer its own DNA because one of the inverted repeats of the piggyback transposon is missing.

  The jellyfish green fluorescent protein gene is obtained by breeding the larva hatched from the silkworm egg and crossing the obtained adult (G0) within the group and observing the fluorescence of the jellyfish green fluorescent protein. Was screened for silkworms that had been introduced into the chromosome. As a result, transgenic silkworms emitting fluorescence by the action of jellyfish green fluorescent protein were obtained.

Example 7. Expression analysis of recombinant protein in silk gland tissue by Western analysis Non-transformed silkworm, transformed silkworm (HP / IC / A transformed silkworm, HP / IC / HA transformed silkworm, HUP / IC / HA) The posterior silk gland tissue of the transformed silkworm) was collected, and the expression of feline interferon ω in the tissue was examined by Western analysis. The posterior silk gland of the silkworm was homogenized in 100 mM sodium phosphate buffer (pH 7.0), and the supernatant was collected after centrifugation. The supernatant was used as a sample, and the cat was collected using ECL PlusTM Western blotting Kit (Amersham Pharmacia) according to the attached protocol. Interferon was detected. That is, the blotted membrane was blocked overnight at 4 ° C. in a blocking solution (5% skim milk / 0.1% Tween 20 / PBS).

  The membrane was washed twice with TPBS (0.1% Tween 20 · PBS) and treated with an anti-feline interferon antibody diluted 1000-fold with TPBS at room temperature for 1 hour. The membrane was washed twice with TPBS and further three times for 5 minutes with TPBS. After that, the mixture was diluted 10,000 times with TPBS, and then treated with an HRP-labeled anti-rabbit IgG antibody for 1 hour at room temperature. After washing the membrane twice with TPBS and further three times for 5 minutes with TPBS, a detection reagent (solution A + solution B) of ECL Plus ™ Western blotting Detection System (manufactured by Amersham Pharmacia) was added. The luminescence was exposed to HyperfilmTM ECLTM and developed. For signal comparison, a molecular imager FXPro manufactured by BIORAD was used.

  As a result, no signal was detected from the posterior silk gland tissue of the untransformed silkworm and the P.IC.A construct-introduced transgenic silkworm, whereas the HP.IC.A construct, the HP.IC.HA construct and the HUP. A signal was detected from the silk gland tissue of the transgenic silkworm transfected with the IC / HA construct. From the results of this experiment, it was found that the region other than the fibroin H chain promoter, that is, the first exon, the first intron, and the second exon region of the fibroin H chain gene, were used to dramatically improve protein synthesis or gene expression in the silkworm gland cells at the rear of the silkworm. Has been reconfirmed that it plays an important role. The result is shown in FIG.

  Accumulated amount of feline interferon in the posterior silk gland tissue of transgenic silkworms containing 5.5kbp of the 5 'promoter region of the fibroin H chain, the HUP / IC / HA gene cassette containing the feline interferon gene and the 3' end of the fibroin H chain Indicates the accumulation of feline interferon in the posterior silk gland tissue of transgenic silkworms containing the 5'-terminal promoter region of the fibroin H chain, the feline interferon gene and the HP / IC / HA gene cassette containing the 3 'end of the fibroin H chain. Was higher. It is thought that there is a gene region in the 5'-terminal upstream region of the H chain that improves the amount of protein produced.

Example 8 Measurement of Recombinant Protein in Silk by Western Analysis Next, secretion of a foreign protein, ie, feline interferon ω into the silk was examined.
We weigh 10 mg each of non-transformed silkworms and transgenic silkworms (HP / IC / A transgenic silkworms, HP / IC / HA transgenic silkworms, HUP / IC / HA transgenic silkworms), After 4 ml of 60% LiSCN was added and stirred, the mixture was allowed to stand at room temperature overnight to dissolve the cocoon. The sample was diluted 10 times with 8M urea, 2% SDS, 5% 2-mercaptoethanol, and the feline interferon was detected using ECL PlusTM Western blotting Kit (Amersham Pharmacia) according to the attached protocol. Was. From the results, the signal intensity was measured using a molecular imager (manufactured by BioRad) and compared with the signal intensity of feline interferon of a known concentration to determine the protein content.

  As a result, no signal was detected from the cocoons of the non-transformed silkworm and the HP / IC / A transgenic silkworm, whereas the HP / IC / HA transgenic silkworm and the HUP / IC / HA transgenic silkworm did not. A signal was detected from the cocoon of the transgenic silkworm, confirming that the feline interferon protein was secreted into the silk thread. The content was about 0.8 to 2.0% for the HP.IC.HA transformed silkworm and about 1.8 to 5.4% for the HUP.IC.HA transformed silkworm. This was 0.4 to 2 mg in terms of weight per silkworm.

  The results of this experiment revealed that the 3′-terminal portion of the fibroin H chain gene plays an important role in the secretion of the protein synthesized in the posterior silk gland cells to the silk. The result is shown in FIG. The amount of feline interferon produced in transgenic silkworms containing the 5 kbp promoter region of the 5 'end of the fibroin H chain, the HUP / IC / HA gene cassette containing the feline interferon gene and the 3' end of the fibroin H chain, is 5% of the fibroin H chain. The production of feline interferon was higher in transgenic silkworms containing the HP / IC / HA gene cassette containing the 'terminal promoter region, the feline interferon gene and the 3' end of the fibroin H chain. It is considered that there is a gene region in the 5'-terminal upstream region of the H chain that improves protein production.

Example 9 Measurement of Recombinant Protein in Silk by ELISA Method Feline interferon ω in silk thread was quantified by ELISA method.
Weigh 10 μg each of non-transformed silkworm and transformed silkworm (HP / IC / A transgenic silkworm, HP / IC / HA transgenic silkworm, HUP / IC / HA transgenic silkworm), After 4 ml of 60% LiSCN was added and stirred, the mixture was allowed to stand at room temperature overnight to dissolve the cocoon. This was diluted 8-fold or 16-fold with PBS and applied to a microtiter plate. As a standard, feline interferon with a known concentration was sequentially diluted with PBS and used.
As a result, feline interferon ω in the silk thread was not detected in the HP / IC / A transgenic silkworm, and was about 1.1 to 2.2% in the HP / IC / HA transgenic silkworm. In the transgenic silkworm, it was about 1.0-4.9%.

  By introducing a gene cassette for expression in which the DNA sequence of the 5′-end portion and the DNA sequence of the 3′-end portion of the fibroin H chain gene are fused to a foreign protein gene into silk gland cells, etc., a large amount of foreign protein can be transferred into the silk gland cells. It has become possible to produce silk glands extracellularly and even silk. By this new method, a foreign protein production technology that can be easily purified was established by using a silk gland to produce a foreign protein without using a recombinant baculovirus.

FIG. 1 is a diagram showing a method (first half) for producing a gene introduction construct containing a PICIC gene cassette. FIG. 2 is a diagram illustrating a method (second half) of preparing a gene-introducing construct containing a P-IC-A gene cassette. FIG. 3 is a diagram showing a method (first half) for producing a gene introduction construct containing an HP / IC / HA gene cassette. FIG. 4 is a diagram showing a method (second half) of preparing a gene introduction construct containing an HP / IC / HA gene cassette. FIG. 5 is a diagram showing a method (first half) of preparing a gene introduction construct containing a HUP / IC / HA gene cassette. FIG. 6 is a diagram showing a method (second half) for preparing a gene introduction construct containing a HUP / IC / HA gene cassette. FIG. 7 is a diagram showing a method (first half) for producing a gene introduction construct containing the HP / IC / A gene cassette. FIG. 8 is a diagram illustrating a method (second half) of preparing a gene introduction construct containing the HP / IC / A gene cassette. FIG. 9 is a diagram in which the expression of β-galactosidase in a cultured silkworm silk gland was analyzed by Western analysis, and is a drawing substitute photograph showing electrophoresis. It was revealed that the first exon, first intron, and second exon region of the fibroin H chain gene play important roles in protein synthesis or gene expression in cells. Extracellular secretion was also confirmed. FIG. 10 is a diagram in which the expression of a recombinant protein in a silkworm silk gland tissue was analyzed by Western analysis, and is a drawing-substituting photograph showing electrophoresis. It was reconfirmed that the fibroin H chain gene first exon, first intron, and second exon region play an important role in dramatically improving the expression of the recombinant protein in the silk gland cells at the rear of the silkworm. In addition, it was revealed that there is a gene region for improving protein production at about 5.5 kbp in the upstream region of the fibroin promoter. FIG. 11 is a diagram in which production of a recombinant protein into a silk thread is analyzed by Western analysis, and is a drawing-substituting photograph showing electrophoresis. It has been clarified that the 3′-terminal portion of the fibroin H chain gene plays an important role in the secretion of the protein synthesized in the silk gland cells into the silk thread. In addition, it was reconfirmed that there is a gene region for improving the amount of protein production at about 5.5 kbp in the upstream region of the fibroin promoter. In addition, it was reconfirmed that a gene region for improving the amount of protein production was present at about 5.5 kbp in the upstream region of the fibroin promoter.

Claims (33)

(1) a promoter expressed in the silk gland;
(2) a gene obtained by fusing the 5 ′ end of the fibroin H chain gene to the 5 ′ side of the foreign protein structural gene, which is linked downstream of (1);
A gene cassette for expression of a foreign protein, comprising: (1) a promoter expressed in the silk gland;
(2) a gene obtained by fusing the 3 ′ end of a fibroin H chain gene to the 3 ′ side of a foreign protein structural gene not containing a termination codon and connected downstream of the above (1);
A gene cassette for expression of a foreign protein comprising: (1) a promoter expressed in the silk gland; and (2) a 3 'end of the 3' end of the fibroin H chain gene, which is connected downstream of (1). A gene cassette for expression of a foreign protein, comprising: a gene obtained by fusing a foreign protein structural gene. (1) a promoter expressed in the silk gland;
(2) The 5 ′ end of the fibroin H chain gene is fused to the 5 ′ side of the foreign protein structural gene not containing a stop codon and downstream of the above (1), and the fibroin is fused to the 3 ′ side of the structural gene. A gene obtained by fusing the 3 'end of the H chain gene;
A gene cassette for expression of a foreign protein, comprising:   The expression of a foreign protein according to claim 1 or 3, wherein the 5 'terminal portion of the fibroin H chain gene contains a part of the first exon, the first intron, and the second exon of the fibroin H chain gene. Gene cassette.   The gene cassette for expression of a foreign protein according to claim 4, wherein the portion of the fibroin in which the first exon and the second exon are combined is a secretory signal gene region of a fibroin H chain gene.   The (1) promoter expressed in the silk gland and (2) the 5′-end portion of the fibroin H chain gene linked downstream of (1) are DNAs shown in SEQ ID NO: 1 or SEQ ID NO: 2. The gene cassette for expression of a foreign protein according to claim 5, characterized in that:   The gene cassette for expressing a foreign protein according to any one of claims 1 to 6, wherein (1) a silk gland promoter and / or (2) 5 ′ of a fibroin H chain gene contained in the gene cassette for expressing a foreign protein. A gene cassette for expression of a foreign protein, characterized by having one or several bases added, deleted or substituted with other bases within a range where the terminal portions have substantially the same function.   The gene cassette for expression of a foreign protein according to any one of claims 1 to 7, wherein the gene cassette for expression of the foreign protein includes (1) a silk gland promoter portion and / or (2) a fibroin H chain gene 5 'A gene cassette for expression of a foreign protein, characterized by having a gene that hybridizes with the portion under stringent conditions in place of the terminal portion and has a gene having substantially the same function as the portion.   The gene cassette for expression of a foreign protein according to claim 2 or 3, wherein the 3 'terminal portion of the fibroin H chain gene contains at least one codon encoding cysteine.   The gene cassette for expression of a foreign protein according to any one of claims 2, 3, and 9, wherein the 3'-terminal portion of the fibroin H chain gene is a DNA represented by SEQ ID NO: 3.   11. The gene cassette for expression of a foreign protein according to claim 10, wherein one or several bases are added or deleted within a range where the 3 ′ terminal portion of the fibroin H chain gene has substantially the same function. Alternatively, a gene cassette for expressing a foreign protein, which has a substitution with another base.   12. The gene cassette for expression of a foreign protein according to claim 10 or 11, wherein the 3 'end of the fibroin H chain gene is hybridized with the 3' end of the fibroin H chain gene under stringent conditions. And a gene cassette for expression of a foreign protein, characterized by having a gene characterized by having substantially the same function.   13. The promoter according to claim 1, wherein the promoter expressed in the silk gland is at least one promoter selected from a promoter of a fibroin H chain gene, a promoter of a fibroin L chain gene, and a promoter of a sericin gene. A gene cassette for expression of a foreign protein according to claim 1.   Downstream of the foreign protein expression gene cassette, at least one polyA additional region selected from a polyA additional region of a fibroin H chain gene, a polyA additional region of a fibroin L chain gene, and a polyA additional region of a sericin gene is provided. The gene cassette for expression of a foreign protein according to any one of claims 1 to 13, which is present.   A chromosome of an insect cell, wherein a pair of piggyBac transposon inverted repeats are present on both sides of the gene cassette for expressing a foreign protein according to any one of claims 1 to 14. Gene cassette for gene transfer into DNA.   An expression vector for insect cells, comprising the gene cassette for expressing a foreign protein according to any one of claims 1 to 14.   A gene transfer vector for an insect cell, comprising the gene cassette for transferring a gene into a chromosome of an insect cell according to claim 15.   18. A method for producing a foreign protein, comprising introducing the insect cell gene transfer vector according to claim 16 or 17 into an insect cell.   19. The method for producing a foreign protein according to claim 18, wherein the insect cell is derived from a Lepidoptera insect.   20. The method for producing a foreign protein according to claim 19, wherein the insect cell is derived from Bombyx mori.   21. The method for producing a foreign protein according to claim 20, wherein the insect cells are silk gland cells of silkworm (Bombyx mori).   Utilizing the DNA transfer activity of piggyback (piggyBac) transposase, a recombinant silkworm in which the gene cassette for expression of the foreign protein was integrated into the chromosome was prepared, and the foreign protein was added to the silk gland or silk of the obtained recombinant silkworm. 19. The method for producing a foreign protein according to claim 18, wherein the foreign protein is recovered from the silk gland or the silk thread after the production.   Simultaneous microinjection of a gene transfer vector for insect cells and DNA or RNA producing piggyback (piggyBac) transposase into silkworm eggs to produce a recombinant silkworm in which the gene cassette for expression of the foreign protein has been integrated into the chromosome. The method for producing a foreign protein according to claim 22, characterized in that:   A recombinant silkworm in which the gene cassette for expressing a foreign protein according to any one of claims 1 to 14 has been introduced into a chromosome, and which has the ability to produce a foreign protein in a silk gland or silk thread.   A silk thread comprising a foreign protein produced by the recombinant silkworm according to claim 24. The following polynucleotide (a) or (b):
(A) a polynucleotide consisting of the nucleotide sequence of position 1-4111 of SEQ ID NO: 2,
(B) a base sequence having one or several bases added, deleted or substituted with other bases in the base sequence (a), wherein the expression of a foreign gene is increased in silk gland tissue; Polynucleotide.   A polynucleotide which hybridizes with a polynucleotide consisting of the nucleotide sequence of position 1-4111 of SEQ ID NO: 2 under stringent conditions, and increases the expression of a foreign gene in silk gland tissue.   A method for increasing the expression of a foreign gene in silk gland tissue, comprising using the polynucleotide according to claim 26 or 27.   A gene cassette for expression of a foreign protein, wherein a promoter of a fibroin H chain gene is linked to the 3 'side of the polynucleotide according to claim 26 or 27, and a foreign protein structural gene is linked to the 3' side thereof.   30. A gene cassette for transferring a gene into the chromosome of an insect cell, wherein a pair of piggyBac transposon inverted repeats are present on both sides of the gene cassette according to claim 29.   31. A recombinant silkworm in which the gene cassette for gene introduction according to claim 30 has been introduced into a chromosome, and which has the ability to produce a recombinant protein in a silk gland or a silk thread.   A silk thread comprising a foreign protein produced by the recombinant silkworm according to claim 31. A silk thread containing a foreign protein (excluding GFP) produced by the recombinant silkworm according to claim 24 or 31.

Images