Sericin gene modification system

Abstract

To provide a silk yarn capable of spinning or an arguloida insect capable of producing the same without heating or using a basic solution.SOLUTION: The present invention discloses an arguloida insect capable of producing a modified sericin protein in which one or more repeating units are deleted in a repeated sequence area and a silk yarn including a modified sericin protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to lepidopteran insects carrying a modified sericin gene, silk threads containing modified sericin proteins, and silk-making methods and peptide extraction methods using them. [Background technology]

[0002] The use of biological materials has attracted more attention as genetic manipulation and genomic information have become easier to use, and is currently expanding. Research and development into such materials is being actively conducted not only in the development of pharmaceuticals using cells, but also in the field of materials.

[0003] Silk thread, which is one of the biological materials, is an ideal material as the basis for developing new materials from the viewpoints of its strength, texture, manufacturing method, processing method, etc. In fact, many studies have been conducted to impart new functions to silk thread (Patent Document 1 and Non-Patent Document 1).

[0004] Conventional silk manufacturing methods include several steps involving heating, such as drying, boiling cocoons, and degumming. In recent years, attempts have been made to add various proteins to silk threads using recombinant gene technology in order to impart new functions to the silk threads. However, problems have arisen in that the added proteins are thermally denatured during the heating process, causing them to lose their functions. There is an increasing need to develop new methods that do not inactivate the added proteins.

[0005] As a method for avoiding heating, a method using a basic solution or an enzyme has been proposed (Patent Document 2). However, although the conditions are milder than those for heat treatment, there are still many proteins that can be denatured by these methods, and the above-mentioned problems have not been completely solved. Therefore, there is still a need to develop a silk production method that does not cause protein denaturation. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2020-70293 A [Patent Document 2] JP 2010-95833 A [Non-patent literature]

[0007] [Non-Patent Document 1] K. Kojima, et al., Biosci. Biotechnol. Biochem. 71, 2943 (2007) Summary of the Invention [Problem to be solved by the invention]

[0008] In the past, attempts have been made to solve the above problems by improving the silk-reeling process, but little has been done to expand the options for silk-reeling processes by modifying the properties of silk itself. If it were possible to modify only the properties of silk that cause heat treatment without affecting its usefulness as a silk material, it would be possible to fundamentally solve the above problems.

[0009] Boiling and degumming are mainly carried out to partially or completely dissolve and remove sericin protein, a paste-like substance in silk thread. Therefore, the silk thread obtained after degumming (degummed thread) does not contain sericin protein, and the sericin protein itself does not significantly affect the usefulness of silk thread as a material. However, since sericin protein is important for cocoon formation as a paste-like component, the loss of sericin protein can inhibit cocoon spinning and reduce the yield of silk thread.

[0010] An object of the present invention is to provide silk thread that can be stably produced without the use of heat or a basic solution, and a lepidopteran insect capable of producing the same. [Means for solving the problem]

[0011] As a result of intensive research conducted by the present inventors to solve the above problems, they have found that by deleting repeating units present in sericin protein, the water solubility of sericin protein can be improved without affecting cocoon spinning. They have also found that silk thread containing this modified sericin protein allows the sericin protein to be eluted without heating. The present invention is based on these novel findings and provides the following:

[0012] [1] A lepidopteran insect capable of producing a modified sericin 1 protein, wherein the gene encoding the modified sericin 1 protein contains all of exon 7 and is missing all or part of exon 6 and / or exon 8. [2] The lepidopteran insect described in [1], wherein the base sequence of the 7th exon has a base sequence having 90% or more of base identity with the base sequence shown in SEQ ID NO: 4, the base sequence of the 6th exon has a base sequence having 90% or more of base identity with the base sequence shown in SEQ ID NO: 3, and the base sequence of the 8th exon has a base sequence having 90% or more of base identity with the base sequence shown in SEQ ID NO: 5. [3] A lepidopteran insect capable of producing a modified sericin 3 protein, wherein a gene encoding the modified sericin 3 protein is missing all or part of a base sequence having 90% or more base identity with the base sequence shown in SEQ ID NO: 15 or a base sequence having 90% or more base identity with the base sequence shown in SEQ ID NO: 16. [4] A lepidopteran insect according to any one of [1] to [3], further comprising an exogenous gene. [5] The lepidopteran insect described in [4], wherein the exogenous gene is linked to a promoter operable in the silk gland. [6] A method for producing raw silk, comprising: a cocoon-spinning step of allowing a lepidopteran insect described in any one of [1] to [5] to form a cocoon; a contacting step of bringing the cocoon into contact with water or an aqueous solution at 4°C to 70°C; a stringing step of stringing the cocoon; and a reeling step of reeling the thread obtained in the stringing step to obtain raw silk. [7] A method for producing a peptide of interest, wherein the peptide of interest is a peptide encoded by an exogenous gene possessed by a lepidopteran insect described in [4] or [5], the method comprising a recovery step of recovering the peptide of interest accumulated in the silk gland lumen from a larva of the lepidopteran insect. [8] A silk thread comprising a modified sericin protein, wherein one or more repeat units are deleted in a repeat sequence region of the modified sericin protein, and the modified sericin protein comprises a modified sericin 1 protein or a modified sericin 3 protein. [9] The silk thread described in [8], wherein the modified sericin 1 protein comprises an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 10, and the repetitive sequence region is at least one of an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 11.

[10] The silk thread described in [9], wherein the repeating unit consists of the amino acid sequence shown in SEQ ID NO: 24 or 45.

[11] The silk thread described in [9] or

[10] , wherein the modified sericin 1 protein is missing all of the amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 9, or all of the amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 11.

[12] The silk thread described in [8], wherein the modified sericin protein comprises a modified sericin 3 protein, and the repeat sequence region is at least one of an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 21.

[13] The silk thread described in

[12] , wherein the repeating unit consists of an amino acid sequence shown in any one of SEQ ID NO: 57 and / or SEQ ID NO: 67 to 70.

[14] The silk thread described in

[12] or

[13] , wherein the modified sericin 3 protein is missing all of the amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 20 or the amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 21.

[15] A method for producing a degummed yarn, comprising a refining step of contacting the silk yarn described in any one of [8] to

[14] with water or an aqueous solution at 4°C to 70°C to degumm it. Effect of the Invention

[0013] According to the lepidopteran insects of the present invention, silk threads having modified sericin proteins can be obtained.

[0014] According to the method for producing raw silk of the present invention, raw silk can be obtained without heat treatment.

[0015] According to the method for producing a target peptide of the present invention, the target peptide can be obtained easily.

[0016] According to the silk thread of the present invention, it is possible to perform spinning without carrying out a heat treatment.

[0017] According to the silk thread of the present invention, a degummed thread can be obtained without carrying out a heat treatment. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a conceptual diagram showing the structure of a silkworm silk gland. A: A conceptual diagram showing a silkworm silk gland. B: A diagram showing the parts that make up a silkworm silk gland and the structure of silk threads in each part. Fibroin produced and secreted in the posterior silk gland is coated on its surface with sericin 1 produced and secreted in the middle and posterior segments of the middle silk gland as it moves from the middle silk gland to the anterior silk gland, and is then further coated with sericin 3 produced and secreted in the anterior segment of the middle silk gland, and is spun from the spinneret. [Diagram 2]Figures showing the structure of the silkworm sericin 1 gene and its splicing. A: A schematic diagram of the silkworm sericin 1 gene (Ser1). B: A schematic diagram showing the structures of splice variants derived from the silkworm Ser1 gene. (a) shows Ser1C, (b) shows Ser1D, (c) shows Ser1B, (d) shows Ser1A, and (e) shows Ser1A'. In figure A, e1 to e9 show the positions of the first to ninth exons, respectively, and i1 to i8 show the positions of the first to eighth introns, respectively, and the double arrows show the location of the repeat sequence region. [Diagram 3] Figures showing the structure of the silkworm sericin 3 gene and its splicing. A: A conceptual diagram of the silkworm sericin 3 gene (Ser3). B: A conceptual diagram showing the structure of a gene spliced ​​from the silkworm Ser3 gene. In figure A, e1 to e3 indicate the positions of the first to third exons, respectively, i1 and i2 indicate the positions of the first and second introns, respectively, and the double arrows indicate the location of the repeat sequence region. [Figure 4] FIG. 1 is a schematic diagram showing the combination of TALEN mRNAs introduced to generate a Ser1 exon 6 deletion line (Ser1 ΔR1) in Example 1. In the figure, e1 to e9 indicate the positions of exon 1 to exon 9, respectively. In the figure, the arrow indicates the recognition site of TALEN mRNA, and the double arrow indicates the location of the repeat sequence region. [Diagram 5] FIG. 1 is a schematic diagram showing the combination of TALEN mRNAs introduced to generate a Ser1 exon 8 deletion line (Ser1 ΔR2) in Example 1. In the figure, e1 to e9 indicate the positions of exon 1 to exon 9, respectively. In the figure, the arrow indicates the recognition site of TALEN mRNA, and the double arrow indicates the location of the repeat sequence region. [Figure 6] FIG. 1 is a schematic diagram showing the combination of TALEN mRNAs introduced to generate a strain lacking the first repeat sequence region of Ser3 (Ser3 ΔR1) in Example 1. In the figure, e1 to e3 indicate the positions of exon 1 to exon 3, respectively. In the figure, the arrow indicates the recognition site of TALEN mRNA, and the double arrow indicates the location of the repeat sequence region. [Figure 7] FIG. 1 is a schematic diagram showing the combination of TALEN mRNAs introduced to generate a strain lacking the second repeat sequence region of Ser3 (Ser3 ΔR2) in Example 1. In the figure, e1 to e3 indicate the positions of the first to third exons, respectively. In the figure, the arrow indicates the recognition site of the TALEN mRNA, and the double arrow indicates the location of the repeat sequence region. [Figure 8] FIG. 1 is a schematic diagram showing the structure of the TALEN mRNA introduced to generate the Ser1 exon 8 deletion strain (Ser1 ΔR1) in Example 1. A: A schematic diagram showing the structure of the vector used for introduction. B: A schematic diagram showing the amino acid sequence of the TALEN to be translated. In the figure, "TAL-N" and "TAL-C" respectively represent the N-terminal domain and C-terminal domain of TALEN, "repeats" represents the repeat sequence for recognizing the target base sequence, and "Fok-I" represents the restriction endonuclease Fok I domain. In FIG. A, "Ampr" represents the ampicillin resistance gene which is a marker gene, "f1 ori" represents the origin of replication of a filamentous bacteriophage, "T7 promoter" represents the T7 promoter recognition sequence for transcription of the TALEN gene, and "SV40 terminator" represents the polyadenylation signal of SV40 (Simian virus 40). In FIG. B, the underlined parts in the repeat sequence represent DNA base recognition sites. [Figure 9] FIG. 1 shows the genetic structure of the Ser1 exon 6 deletion line (Ser1 ΔR1) produced in Example 1 and the results of a confirmation experiment. A: A conceptual diagram showing the genetic structure of Ser1 ΔR1. B: A diagram showing the results of PCR to confirm the presence or absence of partial deletion. C: A diagram showing the results of SDS-PAGE to confirm the expression of the partially deleted sericin protein. In FIG. A, e1 to e9 indicate the positions of exon 1 to exon 9, respectively, the dashed line indicates the deleted region, the thick arrows indicate the positions of the primers used in PCR, and the double arrows indicate the location of the repeat sequence region. Among the thick arrows, the diagonal line arrow indicates a primer that amplifies a fragment only in the wild type (WT), and the white arrow indicates a primer that amplifies a fragment only in the partially deleted type. [Figure 10] FIG. 1 shows the genetic structure of the Ser1 exon 8 deletion line (Ser1 ΔR2) produced in Example 1 and the results of a confirmation experiment. A: A conceptual diagram showing the genetic structure of Ser1 ΔR2. B: A diagram showing the results of PCR to confirm the presence or absence of partial deletion. C: A diagram showing the results of SDS-PAGE to confirm the expression of the partially deleted sericin protein. In FIG. A, e1 to e9 indicate the positions of exon 1 to exon 9, respectively, the dashed line indicates the deleted region, the thick arrows indicate the positions of the primers used in PCR, and the double arrows indicate the location of the repeat sequence region. Among the thick arrows, the diagonal line arrow indicates a primer that amplifies a fragment only in the wild type (WT), and the white arrow indicates a primer that amplifies a fragment only in the partially deleted type. [Figure 11] 1 shows the genetic structure of the line (Ser3 ΔR1) in which the first repeat sequence region of Ser3 has been deleted, produced in Example 1, and the results of a confirmation experiment. A: A conceptual diagram showing the genetic structure of Ser3 ΔR1. B: A diagram showing the results of PCR to confirm the presence or absence of partial deletion. C: A diagram showing the results of SDS-PAGE to confirm the expression of the partially deleted sericin protein. In FIG. A, e1 to e3 indicate the positions of the first to third exons, respectively, the dashed line indicates the deleted region, the white arrow indicates the position of the primers used in PCR, and the double arrow indicates the location of the repeat sequence region. [Figure 12] 1 shows the genetic structure of the line (Ser3 ΔR2) in which the second repeat sequence region of Ser3 has been deleted, produced in Example 1, and the results of a confirmation experiment. A: A conceptual diagram showing the genetic structure of Ser3 ΔR2. B: A diagram showing the results of PCR to confirm the presence or absence of partial deletion. C: A diagram showing the results of SDS-PAGE to confirm the expression of the partially deleted sericin protein. In FIG. A, e1 to e3 indicate the positions of the first to third exons, respectively, the dashed line indicates the deleted region, the white arrow indicates the position of the primers used in PCR, and the double arrow indicates the location of the repeat sequence region. [Figure 13]1 shows the results of SDS-PAGE of the Ser1 exon 6 deletion strain (Ser1 ΔR1) in Example 2. In the figure, "M" indicates the lane in which the size marker was run, and "WT" indicates the results for the wild-type strain. [Figure 14] 1 shows the results of SDS-PAGE of the Ser1 exon 8 deletion strain (Ser1 ΔR2) in Example 2. In the figure, "M" indicates the lane in which the size marker was run, and "WT" indicates the results for the wild-type strain. [Figure 15] 1 shows the results of SDS-PAGE of the strain lacking the first repeat sequence region of Ser3 (Ser3 ΔR1) in Example 2. In the figure, "M" indicates the lane in which the size marker was loaded, and "WT" indicates the results for the wild-type strain. [Figure 16] 1 shows the results of SDS-PAGE of a strain lacking the second repeat sequence region of Ser3 (Ser3 ΔR2) in Example 2. In the figure, "M" indicates the lane in which a size marker was loaded, and "WT" indicates the results for the wild-type strain. [Figure 17] 1 shows the results of the reduction rate of the lines carrying the modified sericin 1 gene (Ser1 ΔR1 and Ser1 ΔR2), in which the error bars indicate standard deviation, and "WT" indicates the results of the wild-type line. [Figure 18] 1 shows the results of the reduction rate of the lines carrying the modified sericin 3 gene (Ser3 ΔR1 and Ser3 ΔR2), in which the error bars indicate standard deviation, and "WT" indicates the results of the wild-type line. [Figure 19] 1 shows the results of the elution rate of sericin from lines carrying the modified sericin 1 gene (Ser1 ΔR1 and Ser1 ΔR2), in which error bars indicate standard deviation and "WT" indicates the results for the wild-type line. [Figure 20] 1 shows the results of the elution rate of sericin from lines carrying the modified sericin 3 gene (Ser3 ΔR1 and Ser3 ΔR2), in which error bars indicate standard deviation and "WT" indicates the results for the wild-type line. [Figure 21]1 shows the results of investigating the relationship between incubation time and reduction rate using a line carrying the modified sericin 1 gene (Ser1 ΔR1), in which error bars indicate standard deviation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] 1. Lepidoptera insects 1-1. Overview The first aspect of the present invention is a lepidopteran insect. The lepidopteran insect of the present invention is a lepidopteran insect capable of producing a modified sericin protein, and capable of spinning silk containing a sericin protein in which one or more repeat units are deleted in the repeat sequence region. Therefore, the insect is capable of spinning cocoons and can be passed on without forming naked pupae.

[0020] 1-2.Definition Key terms used in this specification are defined below.

[0021] "Lepidoptera insects" refers to insects belonging to the taxonomic order Lepidoptera, and refers to butterflies or moths. Butterflies include insects belonging to the families Nymphalidae, Papilionidae, Pieridae, Lycaenidae, and Hesperiidae. Moths include insects belonging to the families Saturniidae, Bombycidae, Brahmaeidae, Eupterotidae, Lasiocampidae, Psychidae, Geometridae, Archtiidae, Noctuidae, Pyralidae, Sphingidae, and the like. For example, moths include species belonging to the genera Bombyx, Samia, Antheraea, Saturnia, Attacus, and Rhodinia, specifically, silkworms, Bombyx mandarina, Samia cynthia (including Samia cynthia ricini and hybrids of Samia cynthia and Samia ricini), Antheraea yamamai, Antheraea pernyi, Saturnia japonica, and Actias gnoma. The lepidopteran insects used in the present invention are not particularly limited, but silkworms, which have high industrial applicability, can be preferably used. Lepidopteran insects in this specification spin silk threads from silk glands.

[0022] In this specification, the term "silk thread" refers to thread derived from lepidopteran insects, and protein thread produced by lepidopteran insect larvae and adults for nesting, moving, fixing, spinning cocoons, capturing food, etc. Silk thread in this specification includes threads derived from any lepidopteran insect. Silk thread is composed of fibroin, a fiber component, and sericin, a water-soluble paste-like component that coats the fibroin. Fibroin is composed of three proteins, fibroin H chain (Fib H), fibroin L chain (Fib L), and p25 / FHX (p25), which form a complex (silk fibroin elementary unit; SFEU complex) in a ratio of Fib H:Fib L:p25=6:6:1. Silk thread in this specification includes any form of silk thread, such as cocoon, raw silk, silk lump, and degummed silk.

[0023] As mentioned above, "fibroin H chain (Fib H)" is one of the three major proteins that make up fibroin, the fiber component of silk thread (Figure 1). It is synthesized in the posterior silk gland, forms an SFEU complex with fibroin L chain and p25, and is then secreted into the lumen of the posterior silk gland.

[0024] As mentioned above, "sericin (Ser)" is a water-soluble gelatin-like protein that is a coating component of silk threads (Figure 1). It is synthesized in the middle silk gland and secreted into the lumen of the middle silk gland. There are several known molecular species of sericin protein that are synthesized from the Ser1, Ser2, and Ser3 genes, but when simply referred to as "sericin" in this specification, it is a general term that includes all molecular species. Three types of sericin are currently known: Ser1, Ser2, and Ser3. Ser1 and Ser3 are found in cocoon threads, and Ser2 is found in scaffold threads during the larval stage.

[0025] As used herein, the term "modified sericin protein" refers to a sericin protein in which one or more repeat units have been deleted in the repeat sequence region through artificial manipulation. In particular, the modified sericin protein used herein refers to modified sericin 1 protein or modified sericin 3 protein.

[0026] As used herein, the term "repeated sequence region" refers to an amino acid sequence region in a sericin protein in which a repeat unit occurs multiple times. The repetitive sequence region herein has complete repeat units at both ends.

[0027] As used herein, the term "repeat unit" refers to the smallest unit of amino acid sequence that appears multiple times in a repeat sequence region. The length and sequence of the repeat unit usually differ depending on the repeat sequence region.

[0028] As used herein, the term "exogenous gene" refers to a foreign gene introduced from the outside through artificial manipulation. The exogenous gene in this specification includes a gene sequence encoding a peptide of interest, and may or may not be expressible by the exogenous gene alone. As used herein, the exogenous gene also includes a gene that exists in a state inserted into a chromosome, such as a progeny of a recombinant organism, so long as its origin is a gene introduced from the outside through artificial manipulation.

[0029] As used herein, the term "target peptide" refers to a peptide encoded by an exogenous gene and to be accumulated in the silk gland of a lepidopteran insect.

[0030] In this specification, the term "peptide" refers to a molecule in which two or more amino acids are linked by an amide bond. Thus, peptides include oligopeptides and polypeptides such as proteins. Peptides include those derived from one gene or a gene fragment thereof, as well as those derived from chimeric genes in which parts of multiple genes are linked together. The amino acid length of a peptide is not particularly limited. For example, the number of amino acid residues may be 10 to 10,000.

[0031] In the present specification, the term "cocoon" refers to a structure consisting of a cocoon layer and an adult, a pupa, an exuvia, and / or a larva. The term "cocoon" in the present specification includes cocoons produced by any lepidopteran insect, so long as the cocoon layer is composed of silk threads.

[0032] As used herein, "raw silk" refers to silk thread reeled from a cocoon and still containing adhesive substances such as sericin. Raw silk as used herein includes not only reeled silk, but also doubled and / or twisted silk.

[0033] As used herein, the term "silk mass" refers to silk threads gathered into a ball of thread. The term "silk mass" as used herein includes, for example, silk floss such as bag floss and square floss.

[0034] In this specification, the term "degummed yarn" refers to spun silk yarn produced from raw silk through degumming. In degummed yarn, some or all of the raw silk that was a bundle of fibers has been converted into single fibers.

[0035] "Reeling" refers to the process of producing raw silk by pulling silk threads from the ends of corded cocoons.

[0036] "Degumming" refers to removing some or all of adhesive substances such as sericin from spun silk threads to obtain single fibers. There are three-tenths, half-tenths, seven-tenths, and full-tenths degumming depending on the degree of degumming, but in this specification, degumming does not limit the degree of degumming or the method used, and broadly refers to any method for removing adhesive substances.

[0037] The term "silk gland" refers to a tubular organ formed by a modified salivary gland, which is an organ of lepidopteran insects that produces, accumulates, and secretes liquid silk. As shown in FIG. 1, silk glands are mainly present in pairs along the digestive tract of silkworm larvae, and each silk gland is composed of three regions: anterior, middle, and posterior silk glands. The anterior silk gland stores single silk filaments in its lumen and secretes them outside the body. The middle silk gland synthesizes sericin, secretes and stores it in its lumen, and secretes it to the anterior silk gland as single silk filaments consisting of fibroin fibers and sericin. The posterior silk gland synthesizes Fib H chain, Fib L chain, and p25, secretes and stores it in its lumen, and secretes it to the middle silk gland as fibroin fibers.

[0038] 1-3.Configuration The lepidopteran insect of the present invention is a lepidopteran insect capable of producing a modified sericin protein. The lepidopteran insect of the present invention comprises a gene encoding a modified sericin 1 protein (hereinafter abbreviated as "modified sericin 1 gene") and / or a gene encoding a modified sericin 3 protein (hereinafter abbreviated as "modified sericin 3 gene"). In an optional configuration, the lepidopteran insect comprises an exogenous gene.

[0039] (1) Modified sericin 1 gene The modified sericin 1 gene encodes a modified sericin 1 protein in which one or more repeat units have been deleted through artificial manipulation in the repeat sequence region of the protein it encodes.

[0040] First, the wild-type sericin 1 gene will be described using the silkworm as an example. The specific base sequence and other factors will vary depending on the type of lepidopteran insect used, but these sequences and methods for correlating them with the silkworm are known in the art.

[0041] As shown in FIG. 2 and SEQ ID NO: 1, the wild-type Ser1 gene of silkworms is composed of nine exons and eight introns (however, the base sequence of the base indicated by n in SEQ ID NO: 1 has not yet been determined, and it is unknown whether it is a, t, g, or c. In addition, sericin 1 has many repetitive sequences, and the number of n is not strict). In addition to the portion indicated by n, it is known that there are individual differences in the base sequence, especially in the intron portion. A specific example of the sequence of the wild-type Ser1 gene is the base sequence shown in SEQ ID NO: 2. The first, second, and seventh to ninth exons are present in all splice variants. In addition, the first and second exons are known to code a signal peptide, and the sixth and eighth exons are known to code the repeated sequence region of the sericin 1 protein. Of these, the third to sixth exons are selected in various combinations by selective splicing. Therefore, many splice variants of sericin 1 protein are known (for example, five splice variants (Ser1A, Ser1A', Ser1B, Ser1C, and Ser1D) are known in the case of silkworms). Normally, the sericin contained in the silk thread spun by lepidopteran insects is a mixture of multiple splice variants, and the properties of each splice variant are unknown.

[0042] The modified sericin 1 gene of the present invention contains the entirety of exon 7, and is missing all or a portion of exon 6 and / or exon 8.

[0043] The nucleotide sequence of each exon is not particularly limited because it differs depending on the Lepidoptera insect. The nucleotide sequence of the wild-type sericin 1 gene of the target Lepidoptera insect and the position of each exon therein can be easily obtained from public sequence databases, etc. For example, in the base sequence shown in SEQ ID NO:2 of the silkworm exemplified above, the first exon is from position 1 to position 93, the second exon is from position 2106 to position 2136, the third exon is from position 5515 to position 6828, the fourth exon is from position 12101 to position 12193, the fifth exon is from position 12513 to position 12590, the sixth exon is from position 13253 to position 19486, the seventh exon is from position 20466 to position 20594, the eighth exon is from position 22520 to position 24118, and the eighth exon is from position 24982 to position 25540.

[0044] The modified sericin 1 gene of the present invention has all of the seventh exon, and examples of the base sequence of the seventh exon include the following sequences: the base sequence shown in SEQ ID NO: 4; a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 4; a base sequence having 90% or more base identity with the base sequence shown in SEQ ID NO: 4; or a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 4.

[0045] In addition, examples of the base sequence of the 6th exon, all or part of which is deleted in the modified sericin 1 gene of the present invention, include the base sequence shown in SEQ ID NO: 3; a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 3; a base sequence having 90% or more base identity with the base sequence shown in SEQ ID NO: 3; or a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 3.

[0046] In addition, examples of the base sequence of exon 8, all or part of which is deleted in the modified sericin 1 gene of the present invention, include the base sequence shown in SEQ ID NO:5; a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO:5; a base sequence having 90% or more base identity with the base sequence shown in SEQ ID NO:5; or a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO:5.

[0047] Here, in this specification, "plurality" refers to 2 to 10, for example, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.

[0048] In this specification, the term "base sequence identity" refers to a numerical value indicating the proportion of sites in which the type of base is the same within the comparison range of two base sequences, similar to the amino acid sequence identity. The base sequence identity can be calculated by aligning the two base sequences so that the base match degree within the comparison range is the highest, even if the lengths of the two base sequences are different. In addition to the above-mentioned BLAST and FASTA, it is also possible to analyze the base sequence identity using an analysis algorithm such as MUMmer, without being limited thereto. The degree of sequence identity here is not particularly limited as long as it is 90% or more. For example, it may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0049] In the present specification, the "high stringent conditions" refer to environmental conditions in which non-specific hybridization is unlikely to occur. Under high stringent conditions, a hybrid can be formed with a nucleic acid having a target base sequence, but a hybrid cannot be substantially formed with a nucleic acid having a non-specific base sequence. Generally, high stringent conditions refer to conditions of low salt concentration and high temperature. A low salt concentration is, for example, 15 mM to 750 mM, preferably 15 mM to 500 mM, 15 mM to 300 mM, or 15 mM to 200 mM. In addition, a high temperature is, for example, 50 to 68°C, or 55 to 70°C. A specific example of a high stringent condition is a condition in which washing after hybridization is performed at 65°C with 0.1×SSC and 0.1% SDS.

[0050] The modified sericin 1 gene of the present invention may be any gene that encodes the modified sericin 1 protein described in the second embodiment, and the specific manner of deletion and the specific base sequence thereof are not particularly limited.

[0051] In the present invention, when a part of exon 6 or 8 is deleted in the modified sericin 1 gene, the length of each deleted region is not particularly limited. For example, the deletion can be performed so that one or more repeat units of the modified sericin 1 protein is reduced. Specifically, for example, 1 base or more, 2 bases or more, 3 bases or more, 5 bases or more, 9 bases or more, 10 bases or more, 20 bases or more, 21 bases or more, 30 bases or more, 50 bases or more, 51 bases or more, 70 bases or more, 72 bases or more, 90 bases or more, 100 bases or more, 102 bases or more, 110 bases or more, 114 bases or more, 120 bases or more, 150 bases or more, 210 bases or more, 500 bases or more, 1000 bases or more, 1200 bases or more, 1500 bases or more, 1599 bases or more, 1600 bases or more, 2000 bases or more, 3000 bases or more, 4000 bases or more, 5000 bases or more, 6000 bases or more, 6100 bases or more, 6200 bases or more, or 6234 bases or more can be deleted.

[0052] The deletion may result in a frameshift, but is preferably made in such a way that no frameshift occurs (ie, the codons are in frame).

[0053] The modified sericin 1 gene of the present invention, for example, lacks the entirety of exon 6 and / or the entirety of exon 8.

[0054] The total length of the deleted region in the modified sericin 1 gene of the present invention is not particularly limited, and may be, for example, 1 base or more, 5 bases or more, 10 bases or more, 20 bases or more, 50 bases or more, 70 bases or more, 90 bases or more, 100 bases or more, 120 bases or more, 150 bases or more, 200 bases or more, 500 bases or more, 1000 bases or more, 1200 bases or more, 1500 bases or more, 1599 bases or more, 1600 bases or more, 1700 bases or more, 2000 bases or more, 2100 bases or more, 2200 bases or more, 2300 bases or more, 2382 bases or more, 2400 bases or more, 250 bases or more, or 300 bases or more. 00 bases or more, 3000 bases or more, 4000 bases or more, 5000 bases or more, 6000 bases or more, 6100 bases or more, 6200 bases or more, 6234 bases or more, 6500 bases or more, 7000 bases or more, 8000 bases or more, 9000 bases or more, 10000 bases or more, 11000 bases or more, 12000 bases or more, 13000 bases or more, 14000 bases or more, 14300 bases or more, 14400 bases or more, 14500 bases or more, or 14510 bases or more can be deleted. The length of the deleted region may be, for example, 25,411 bases or less, 25,000 bases or less, 24,000 bases or less, 23,000 bases or less, 22,000 bases or less, 21,000 bases or less, 20,000 bases or less, 19,000 bases or less, 18,000 bases or less, 17,000 bases or less, 16,000 bases or less, 15,500 bases or less, 15,400 bases or less, 15,300 bases or less, 15,200 bases or less, 15,100 bases or less, 15,000 bases or less, 14,600 bases or less, 14,510 bases or less, 15,200 bases or less, 15,300 bases or less, 15,400 bases or less, 15,400 bases or less, 15,500 bases or less, 15,600 bases or less, 15,700 bases or less, 15,800 bases or less, 15,900 bases or less, 15,100 bases or less, 15,000 bases or less, 14,600 bases or less, 14,51 ...100 bases or less, 15,200 bases or less, 15,300 The length can be 14,500 bases or less, 14,400 bases or less, 14,000 bases or less, 13,000 bases or less, 12,000 bases or less, 11,000 bases or less, 10,000 bases or less, 9,000 bases or less, 8,000 bases or less, 7,000 bases or less, 6,500 bases or less, 6,234 bases or less, 6,200 bases or less, 6,000 bases or less, 5,000 bases or less, 4,000 bases or less, 3,000 bases or less, 2,500 bases or less, 2,400 bases or less, 2,382 bases or less, etc.

[0055] The nucleotide sequence of the deleted region in the modified sericin 1 gene of the present invention is not particularly limited. It may consist of only an exon nucleotide sequence, or may contain an exon nucleotide sequence and an intron nucleotide sequence. In addition, for example, both or one ends of the deleted region may contain an intron nucleotide sequence. Preferably, both ends of the deleted region contain an intron nucleotide sequence.

[0056] When both or one of the ends of the deleted region contains an intron nucleotide sequence, the length of the intron nucleotide sequence is not particularly limited, for example, 50 bases or more, 60 bases or more, 70 bases or more, 80 bases or more, 90 bases or more, 100 bases or more, 110 bases or more, 120 bases or more, 130 bases or more, 140 bases or more, 145 bases or more, 150 bases or more, 155 bases or more, 160 bases or more, 190 bases or more, 200 bases or more, 210 bases or more, 215 bases or more, 220 bases or more, 224 bases or more, 225 bases or more, 230 bases or more, 240 bases or more, 250 bases or more, 270 bases or more, It can be 290 bases or more, 300 bases or more, 350 bases or more, 370 bases or more, 380 bases or more, 385 bases or more, 390 bases or more, 400 bases or more, 450 bases or more, 500 bases or more, 520 bases or more, 530 bases or more, 535 bases or more, 538 bases or more, 540 bases or more, 550 bases or more, 600 bases or more, 610 bases or more, 620 bases or more, 624 bases or more, 625 bases or more, 630 bases or more, or 631 bases or more. For example, when it contains an intron base sequence at both ends, each can be, for example, 50 bases to 630 bases, 100 bases to 630 bases, 140 bases to 630 bases, 120 bases to 500 bases, 140 bases to 400 bases, etc.

[0057] The modified sericin 1 gene of the present invention may additionally contain any other mutation, and the type and manner of the mutation are not particularly limited. For example, any sequence region may be added, deleted and / or substituted. The type of other exons contained in the modified sericin 1 gene is not particularly limited. The modified sericin 1 gene of the present invention may contain one or more additional exons in any combination, or may contain no additional exons.

[0058] When no additional exons are included, the modified sericin 1 gene includes only exon 7; only all or a portion of exon 7 and exon 6; only all or a portion of exon 7 and exon 8; only all or a portion of exon 7, exon 6, and a portion of exon 8; or only exon 7, a portion of exon 6, and a portion of exon 8.

[0059] When the modified sericin 1 gene contains an additional exon, the additional exon preferably contains all or a part of one or more of exon 1, exon 2, and exon 9. Specifically, for example, the modified sericin 1 gene contains all or a part of exon 1, exon 2, and exon 9 as the additional exon. Also, for example, the modified sericin 1 gene contains all or a part of exon 1, exon 2, and exon 9 as the additional exon, as well as all or a part of exon 3, exon 4, and exon 5 as the additional exon.

[0060] The modified sericin 1 gene preferably exhibits expression similar to that of the wild-type sericin 1 gene, and the lepidopteran insect, for example, contains the modified sericin 1 gene in place of the wild-type sericin 1 gene or controlled by the regulatory sequence of the wild-type sericin 1 gene.

[0061] The specific base sequence of the modified sericin 1 gene is not particularly limited, and examples thereof include the base sequence shown in SEQ ID NO: 6 or 7, a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 6 or 7, a base sequence having 90% or more base identity with the base sequence shown in SEQ ID NO: 6 or 7, or a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 6 or 7.

[0062] The lepidopteran insect of this embodiment contains a homozygous or heterozygous modified sericin 1 gene. For example, when all of the sericin 1 genes contained in the lepidopteran insect are modified sericin 1 genes, all of the sericin 1 genes may be modified by the same modification, or each of the sericin 1 genes may be modified by different gene modifications.

[0063] (2) Modified sericin 3 gene The modified sericin 3 (Ser3) gene encodes a sericin 3 protein in which one or more repeat units have been deleted through artificial manipulation in the repeat sequence region of the protein it encodes.

[0064] First, the wild-type Ser3 gene will be described using the silkworm as an example. The specific base sequence and other information will vary depending on the type of lepidopteran insect used, but these sequences and methods for correlating them with the silkworm are known in the art.

[0065] The wild-type Ser3 gene of silkworm consists of three exons and two introns, as shown in Figure 3 and SEQ ID NO: 14. There are no splice variants in sericin 3, and all sericin 3 contains the nucleotide sequences of exons 1, 2 and 3. Among these, exon 3 contains a nucleotide sequence encoding two repeat sequence regions.

[0066] The modified sericin 3 gene of the present invention lacks all or part of either or both of the nucleotide sequences encoding the two repeat sequence regions of the third exon.

[0067] The base sequence of each exon is not particularly limited, since it differs depending on the Lepidoptera insect. The base sequence of the wild-type sericin 2 gene of the target Lepidoptera insect and the position of each exon therein can be easily obtained from a public sequence database, etc. For example, in the base sequence of the silkworm shown in SEQ ID NO: 14 exemplified above, the first exon is at positions 1 to 33, the second exon is at positions 1344 to 1374, and the third exon is at positions 2491 to 6242.

[0068] Examples of the base sequence of the third exon, at least a portion of which is deleted in the modified sericin 3 gene of the present invention, include the base sequence shown in SEQ ID NO: 111; a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 111; a base sequence having 90% or more base identity with the base sequence shown in SEQ ID NO: 111; or a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 111.

[0069] Examples of the base sequences of the two repeat sequence regions contained in the base sequence of the third exon include the base sequences shown in SEQ ID NOs: 15 and 16; base sequences in which one or more bases have been added, deleted, and / or substituted in the base sequences shown in SEQ ID NOs: 15 and 16; base sequences having 90% or more base identity with the base sequences shown in SEQ ID NOs: 15 and 16; or base sequences that hybridize under highly stringent conditions to base sequences complementary to the base sequences shown in SEQ ID NOs: 15 and 16. For example, the modified sericin 3 gene lacks all or a part of the base sequence having 90% or more base identity with the base sequence shown in SEQ ID NO: 15 or the base sequence having 90% or more base identity with the base sequence shown in SEQ ID NO: 16.

[0070] When the modified sericin 3 gene of the present invention has a partial deletion of a repeat sequence region, the length of each deleted region is not particularly limited. For example, the deletion can be performed so that one or more repeat units of the modified sericin 3 protein are reduced. Specifically, for example, 1 base or more, 2 bases or more, 3 bases or more, 5 bases or more, 9 bases or more, 10 bases or more, 20 bases or more, 21 bases or more, 30 bases or more, 50 bases or more, 51 bases or more, 70 bases or more, 72 bases or more, 90 bases or more, 100 bases or more, 102 bases or more, 110 bases or more, 114 bases or more, 120 bases or more, 150 bases or more, 200 bases or more, 400 bases or more, 420 bases or more, 440 bases or more, 444 bases or more, 445 bases or more, 450 bases or more, 460 bases or more, 500 bases or more, 540 bases or more, 550 bases or more, 555 bases or more, 558 bases or more, 560 bases or more, 570 bases or more, 6 00 bases or more, 650 bases or more, 700 bases or more, 710 bases or more, 720 bases or more, 725 bases or more, 730 bases or more, 750 bases or more, 1000 bases or more, 1200 bases or more, 1500 bases or more, 2000 bases or more, 2500 bases or more, 2510 bases or more, 2514 bases or more, 2515 bases or more, 2520 bases or more, 2550 bases or more, 2600 bases or more, 2610 bases or more, 2620 bases or more, 2625 bases or more, 2630 bases or more, 2700 bases or more, 2750 bases or more, 2800 bases or more, 2850 bases or more, 2870 bases or more, 2900 bases or more, or 3000 bases or more can be deleted.

[0071] The deletion may result in a frameshift, but is preferably made in such a way that no frameshift occurs (ie, the codons are in frame).

[0072] The modified sericin 3 gene of the present invention, for example, lacks all of two repeat sequence regions or all of one repeat sequence region.

[0073] The total length of the deleted region in the modified sericin 3 gene of the present invention is not particularly limited, and may be, for example, 1 base or more, 5 bases or more, 10 bases or more, 20 bases or more, 50 bases or more, 70 bases or more, 90 bases or more, 100 bases or more, 120 bases or more, 150 bases or more, 200 bases or more, 400 bases or more, 420 bases or more, 440 bases or more, 444 bases or more, 445 bases or more, 450 bases or more, 460 bases or more, 500 bases or more, 540 bases or more, 550 bases or more, 555 bases or more, 558 bases or more, 560 bases or more, 570 bases or more, 600 bases or more, 650 bases or more, 700 bases or more, 710 bases or more, 720 bases or more, 725 bases or more, 730 bases or more, 740 bases or more, 750 bases or more, 760 bases or more, 770 bases or more, 780 bases or more, 790 bases or more, 800 bases or more, 810 bases or more, 820 bases or more, 830 bases or more, 840 bases or more, 850 bases or more, 860 bases or more, 870 bases or more, 880 bases or more, 890 bases or more, 900 bases or more, 910 bases or more, 920 bases or more, 930 bases or more, 940 bases or more, 950 bases or more, 96 30 bases or more, 750 bases or more, 1000 bases or more, 1200 bases or more, 1500 bases or more, 2000 bases or more, 2500 bases or more, 2510 bases or more, 2514 bases or more, 2515 bases or more, 2520 bases or more, 2550 bases or more, 2600 bases or more, 2610 bases or more, 2620 bases or more, 2625 bases or more, 2630 bases or more, 2700 bases or more, 2750 bases or more, 2800 bases or more, 2850 bases or more, 2870 bases or more, 2900 bases or more, 3000 bases or more, 3500 bases or more, 4000 bases or more, or 4500 bases or more can be deleted. The length of the deleted region can be, for example, 8000 bases or less, 7000 bases or less, 6500 bases or less, 6300 bases or less, 6250 bases or less, 6242 bases or less, 6200 bases or less, 6000 bases or less, 5000 bases or less, 4500 bases or less, 4000 bases or less, 3500 bases or less, 3000 bases or less, 2700 bases or less, 2650 bases or less, 2625 bases or less, 2620 bases or less, 2600 bases or less, 2500 bases or less, 2000 bases or less, 1500 bases or less, 1000 bases or less, 800 bases or less, 600 bases or less, 560 bases or less, 558 bases or less, etc.

[0074] The base sequence of the deleted region in the modified sericin 3 gene of the present invention is not particularly limited. It may consist of only the base sequence of an exon, or may contain the base sequence of an exon and the base sequence of an intron. In addition, for example, both or one end of the deleted region may contain the base sequence of an intron. In addition, for example, even if the deleted region consists of only the base sequence of an exon, both or one end of the deleted region may contain an extra base sequence.

[0075] When both or one end of the deleted region contains an extra base sequence other than the repeat sequence region, the length of the extra base sequence is not particularly limited. For example, it can be 1 base or more, 10 bases or more, 20 bases or more, 30 bases or more, 31 bases or more, 32 bases or more, 33 bases or more, 34 bases or more, 35 bases or more, 36 bases or more, 40 bases or more, 50 bases or more, 60 bases or more, 70 bases or more, 75 bases or more, 76 bases or more, 77 bases or more, 78 bases or more, 79 bases or more, 80 bases or more, 90 bases or more, 100 bases or more, 105 bases or more, 109 bases or more, 110 bases or more, or 114 bases or more. For example, when an extra base sequence is contained at both ends, each can be, for example, 10 bases to 100 bases, 20 bases to 90 bases, 30 bases to 80 bases, etc.

[0076] When one or both ends of the deleted region contain an intron nucleotide sequence, the length of the intron nucleotide sequence is not particularly limited and may be within the range described above for the modified sericin 1 gene.

[0077] Furthermore, the deleted region may contain, for example, the base sequence of an exon other than the third exon, or may contain only the base sequence of the third exon as the exon base sequence.

[0078] The modified sericin 3 gene preferably exhibits expression similar to that of the wild-type sericin 3 gene, and the lepidopteran insect, for example, contains the modified sericin 3 gene in place of the wild-type sericin 3 gene or controlled by the regulatory sequence of the wild-type sericin 3 gene.

[0079] The specific base sequence of the modified sericin 3 gene is not particularly limited, and examples thereof include the base sequence shown in SEQ ID NO: 17 or 18, a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 17 or 18, a base sequence having 90% or more base identity with the base sequence shown in SEQ ID NO: 17 or 18, or a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 17 or 18.

[0080] The lepidopteran insect of this embodiment contains a homozygous or heterozygous modified sericin 3 gene. For example, when the sericin 3 genes contained in the lepidopteran insect are all modified sericin 3 genes, all of the sericin 3 genes may be modified by the same modification, or each of the sericin 3 genes may be modified by different gene modifications.

[0081] Furthermore, the lepidopteran insect of this embodiment may contain both the modified sericin 1 gene and the modified sericin 3 protein. In this case, the modified genes may be homozygous, or at least one of the modified genes may be heterozygous. For example, when all sericin genes possessed by the lepidopteran insect are modified genes, all copies of the genes may be differently modified genes.

[0082] (3) Exogenous genes The lepidopteran insect of this embodiment can further comprise an exogenous gene. The type of exogenous gene possessed by the lepidopteran insect is not particularly limited. The silk obtained from the lepidopteran insect of the present invention can be treated under conditions that make it difficult for the target peptide encoded by the exogenous gene to denature. Therefore, as the target peptide encoded by the exogenous gene, a peptide and / or protein that is easily denatured by heat and / or degumming enzymes, etc., can be used.

[0083] In the present specification, the exogenous gene includes both natural genes and artificial genes. The organism from which the exogenous gene is derived is not particularly limited. For example, the exogenous gene may be a gene possessed by the lepidopteran insect or a gene derived from another organism. The type of the exogenous gene is not particularly limited. For example, the exogenous gene may be a gene not derived from silk thread, a gene derived from silk thread, or a combination thereof.

[0084] The specific type of exogenous gene is not particularly limited, and may be, for example, a gene encoding a peptide exemplified as the peptide of interest in the second embodiment. If necessary, the exogenous gene may be codon-optimized for expression in the lepidopteran insect used. The specific method is not particularly limited, and any method known in the art may be used.

[0085] The exogenous gene is preferably constructed so as to be expressible in the silk gland under certain conditions. In this case, the part of the silk gland in which the gene can be expressed is not particularly limited, and may include, for example, the middle silk gland and / or the posterior silk gland.

[0086] The exogenous gene is preferably linked to a promoter that can operate under a certain control. In this case, the type of the promoter is not particularly limited.

[0087] As used herein, a "promoter" refers to a gene expression regulatory region capable of controlling the expression of a gene or the like located downstream (on the 3' end side). When classified based on the location at which a gene or the like under expression control is expressed, promoters can be divided into ubiquitous promoters (systemic promoters) and site-specific promoters. A ubiquitous promoter is a promoter that controls the expression of a target gene or the like (a target gene or the like) in all cells, i.e., the entire host individual. A site-specific promoter is a promoter that controls the expression of a target gene or the like only in a specific cell or tissue. The promoter that controls an exogenous gene of the present invention may be either a ubiquitous promoter or a site-specific promoter, but it is preferable that the promoter be capable of inducing expression in the silk gland.

[0088] When classified based on the time of expression, promoters are classified into constitutively active promoters, expression-inducible promoters, and time-specifically active promoters. A constitutively active promoter can constitutively express a target gene, etc., in a cell. An expression-inducible promoter can induce the expression of a target gene, etc., in a cell at any time based on an inductive stimulus. Furthermore, a time-specifically active promoter can induce the expression of a target gene, etc., in a cell only at a specific time during the developmental stage. The promoter that controls the exogenous gene of the present invention may be a constitutively active promoter, expression-inducible promoter, or time-specifically active promoter, but it is preferable that it can induce expression in the silk gland.

[0089] The exogenous gene is preferably linked to a promoter operable in the silk gland, and in particular, is preferably linked to, for example, an expression-inducible promoter and / or a site-specific promoter.

[0090] In the case of an expression-inducible promoter, it is preferable that the expression is induced based on the presence of a protein expressed in the silk gland of the target. In this case, the type of protein capable of inducing the expression of an exogenous gene is not particularly limited. For example, it may be an endogenous protein of a lepidopteran insect, or an exogenous protein introduced from the outside. In particular, when an exogenous protein is used, for example, a lepidopteran insect strain carrying a gene encoding an exogenous protein linked to a site-specific promoter and a lepidopteran insect of this embodiment can be crossed to obtain a lepidopteran insect capable of spinning silk of the second embodiment.

[0091] In the case of a site-specific promoter, for example, a promoter capable of being expressed specifically in a site including the silk gland of the target can be used. As such a promoter, a promoter of a gene expressed in the silk gland of the target, such as a promoter of a sericin gene, can be used.

[0092] 1-4.Creation method As long as a lepidopteran insect having the above-mentioned genetic constitution can be obtained, the method for producing the same is not particularly limited. Hereinafter, the method for producing a lepidopteran insect of this embodiment will be described by way of example using a silkworm as an example, but an appropriate method can be selected depending on the type of lepidopteran insect used and the specific region to be deleted.

[0093] (1) Modification of the sericin gene Deletion of exon 6 and / or exon 8 in the wild-type Ser1 gene of a lepidopteran insect, and / or deletion of the repeat unit in the Ser3 gene, can be carried out using any genome editing technique known in the art.

[0094] The technique used is not particularly limited. For example, when a lepidopteran insect contains multiple deletions, each deletion may be performed using a different technique, or one or more regions may be deleted using a common technique.

[0095] In this specification, "genome editing" refers to a gene targeting technique that uses a DNA repair mechanism associated with double strand break (DSB) caused by a DNA cleavage enzyme to insert a foreign gene (knock-in) or destroy a target gene (knock-out) at any position on the genome. Known genome editing techniques include the zinc finger nuclease (ZFN) method, the TALEN method, and the CRISPR / Cas method, and any of these methods may be used in this specification.

[0096] The "TALEN (Transcription Activator-Like Effector Nuclease) method" is a genome editing technique using an artificial DNA cleavage enzyme that fuses a TAL effector (TALE) protein derived from the plant pathogenic bacterium Xanthomonas with a non-specific endonuclease domain. A gene knockout method using the TALEN method in lepidopteran insects such as silkworms is a well-known technique. For example, the method described in Takasu Y., et al., 2013, PLoS One 8, e73458 may be used as a reference.

[0097] The "Zinc Finger Nuclease (ZFN) method" is a genome editing technology that uses an artificial DNA cleaving enzyme consisting of a zinc finger domain as a DNA binding domain and a non-specific endonuclease domain such as the nuclease domain of FokI. Since one zinc finger motif can recognize three bases and bind to a target nucleic acid, by linking multiple zinc finger motifs, it specifically recognizes and binds to three times the number of bases linked. It functions as a dimer, and after binding to the target site, it uses its endonuclease activity to create a double-strand break (DSB) at a specific site in the target nucleic acid.

[0098] The "CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR associated proteins) method" is a genome editing technology that utilizes the adaptive immune system that evolved in bacteria and archaea to eliminate foreign DNA or RNA such as viruses and plasmids. In addition to the CRISPR / Cas9 method that uses the Cas9 protein, variations using other Cas proteins such as Cpf1 and Cas13a have been reported.

[0099] As long as the above-mentioned modified sericin gene is formed, the specific cleavage position of the genome is not particularly limited. For example, in the case of the sericin 1 gene, positions 5300 to 5400, 19800 to 19900, 21850 to 21950, and 24200 to 24300 in SEQ ID NO: 2 can be used as cleavage positions. In addition, for example, in the case of the sericin 3 gene, positions 2800 to 2900, 5400 to 5500, and 5950 to 6050 in SEQ ID NO: 14 can be used as cleavage positions.

[0100] (2) Generation of homozygous individuals of the repeat unit deletion line In the silkworms into which the modified sericin gene was introduced in (1), the modified sericin gene is heterozygous, so that homozygous individuals for the modified sericin gene may be obtained, if necessary. Homozygous individuals can be obtained by inbreeding or brother-sister mating, and the desired homozygous individuals can be selected based on index traits. In this specification, "inbreeding" refers to mating between individuals that have the same target mutant gene (here, the modified sericin gene). Furthermore, "brother-sister mating" refers to inbreeding between males and females of the same litter.

[0101] Furthermore, if necessary, silkworms having multiple types of modified sericin genes can be crossbred with individuals from multiple strains containing different modified sericin genes to obtain silkworms having multiple types of modified sericin genes.

[0102] Effects The lepidopteran insect of this embodiment can spin silk containing modified sericin 1 protein and / or modified sericin 3 protein with reduced repeat units, which facilitates the removal of sericin in the subsequent silk-reeling process.

[0103] By adopting the above-mentioned constitution, the lepidopteran insect of this embodiment can produce sericin that has improved water solubility but retains its function as a paste-like component, which allows the lepidopteran insect of this embodiment to spin cocoons and facilitates lineage maintenance by successive generations.

[0104] 2. Silk thread 2-1. Overview A second aspect of the present invention is a silk thread comprising a modified sericin protein in which one or more repeat units are deleted in the repeat sequence region.

[0105] 2-2.Configuration The silk thread of the present invention comprises a modified sericin protein having one or more repeat units deleted in its repeat sequence region, where the modified sericin protein comprises a modified sericin 1 protein or a modified sericin 3 protein. Optionally, the silk thread of this embodiment comprises a peptide of interest.

[0106] (1) Modified sericin 1 protein The modified sericin 1 protein is a sericin 1 protein in which one or more repeat units are deleted in the repeat sequence region present in the amino acid region encoded by exon 6 or exon 8. For example, a wild-type silkworm sericin 1 protein includes the amino acid sequence shown in SEQ ID NO:8.

[0107] The modified sericin 1 protein preferably contains an amino acid region encoded by exon 7. Examples of the amino acid sequence of the amino acid region encoded by exon 7 include the amino acid sequence shown in SEQ ID NO: 10; an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 10; or an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 10. Note that the first base of the codon for the amino acid at position 1 in the amino acid sequence shown in SEQ ID NO: 10 is derived from exon 6, and therefore the amino acid at position 1 in the amino acid sequence shown in SEQ ID NO: 10 may be substituted with a different amino acid.

[0108] Here, the amino acid substitution is preferably a conservative amino acid substitution. In general, if the amino acid substitution is conservative, the protein having the amino acid sequence after the substitution is likely to have substantially the same structure or properties as the amino acid sequence before the substitution. Conservative amino acids refer to the relationship between amino acids classified into the same amino acid group. The amino acid groups include nonpolar amino acids (glycine, alanine, phenylalanine, valine, leucine, isoleucine, methionine, proline, tryptophan), polar amino acids (amino acids other than nonpolar amino acids), charged amino acids (acidic amino acids (aspartic acid, glutamic acid) and basic amino acids (arginine, histidine, lysine)), uncharged amino acids (amino acids other than charged amino acids), aromatic amino acids (phenylalanine, tryptophan, tyrosine), branched chain amino acids (leucine, isoleucine, valine), and aliphatic amino acids (glycine, alanine, leucine, isoleucine, valine).

[0109] As used herein, the term "amino acid identity" refers to the percentage (%) of identical amino acids in one polypeptide relative to the total number of amino acids in the other polypeptide when the amino acid sequences of two polypeptides are aligned and, if necessary, gaps are introduced into one of the amino acid sequences to maximize the degree of amino acid identity between the two polypeptides. The percentage of amino acid identity can be easily determined by using a known program such as the homology search program BLAST (Basic local alignment search tool; Altschul, SF et al., J. Mol. Biol., 215, 403-410, 1990). The degree of sequence identity here is not particularly limited as long as it is 90% or more. For example, it may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0110] Examples of the amino acid sequence of the repeat sequence region encoded by the 6th exon include the amino acid sequence shown in SEQ ID NO: 9; an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 9; or an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 9. Note that since the first base of the codon for the amino acid at position 1 in the amino acid sequence shown in SEQ ID NO: 9 is derived from the 5th exon, in this respect the amino acid at position 1 in the amino acid sequence shown in SEQ ID NO: 9 may be substituted with a different amino acid.

[0111] Examples of the amino acid sequence of the repeat sequence region encoded by exon 8 include the amino acid sequence shown in SEQ ID NO: 11, an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 11. Note that since the first base of the codon for the amino acid at position 1 in the amino acid sequence shown in SEQ ID NO: 11 is derived from exon 7, in this regard, the amino acid at position 1 in the amino acid sequence shown in SEQ ID NO: 11 may be substituted with a different amino acid.

[0112] The repeat sequence region from which the repeat unit is deleted in the modified sericin 1 protein is preferably at least one of the repeat sequence regions encoded by exon 6 or exon 8. For example, the repeat unit may be deleted only in either the repeat sequence region encoded by exon 6 or exon 8, or the repeat unit may be deleted in both of the repeat sequence regions.

[0113] The amino acid sequence of the repeat unit in the repeat sequence region encoded by exon 6 or exon 8 is composed of 38 amino acids. The amino acid sequence of the repeat unit is not particularly limited as long as it is an amino acid sequence composed of 38 amino acids and is repeated with a certain sequence identity. For example, specific amino acid sequences of the repeat unit include an amino acid sequence consisting of the consensus sequence shown in SEQ ID NO: 24 for exon 6 and SEQ ID NO: 45 for exon 8; an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 24 or 45; or an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 24 or 45.

[0114] More specifically, examples of the amino acid sequence of the repeating unit of the 6th exon include an amino acid sequence consisting of an amino acid sequence shown in any one of SEQ ID NOs: 24 to 44; an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in any one of SEQ ID NOs: 24 to 44; and an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in any one of SEQ ID NOs: 24 to 44.

[0115] Examples of the amino acid sequence of the repeating unit of exon 8 include an amino acid sequence consisting of an amino acid sequence shown in any one of SEQ ID NOs: 46 to 56; an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in any one of SEQ ID NOs: 46 to 56; and an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in any one of SEQ ID NOs: 46 to 56.

[0116] The number of repeat units deleted is not particularly limited as long as the number of complete repeat units is reduced. The repeat sequence region encoded by exon 6 usually has 53 repeat units. On the other hand, the repeat sequence region encoded by exon 8 usually has 13 repeat units. Specific numbers of repeat units deleted in the modified sericin 1 protein include, for example, 1 or more, 2 or more, 3 or more, 5 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 51 or more, 52 or more, 53 or more, 55 or more, 60 or more, 65 or more, and 66. In addition, for example, in the modified sericin 1 protein, 66 or less, 65 or less, 60 or less, 55 or less, 53 or less, 52 or less, 51 or less, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, 14 or less, or 13 or less repeat units can be deleted.

[0117] In addition, the number of complete repeat units may be reduced by, for example, deleting a part of the repeat units, and the number of complete repeat units to be reduced is not particularly limited. Specifically, for example, the number of complete repeat units to be deleted may be reduced by the number exemplified above.

[0118] The specific deletion mode of the entire modified sericin 1 protein is not particularly limited. For example, the modified sericin 1 protein may be a modified sericin 1 protein lacking the entire amino acid sequence encoded by the 6th exon (e.g., an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO:9) and / or the entire amino acid sequence encoded by the 8th exon (e.g., an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO:11). Further, the modified sericin 1 protein may be a modified sericin 1 protein lacking either the entire amino acid sequence encoded by the 6th exon (e.g., an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO:9) or the entire amino acid sequence encoded by the 8th exon (e.g., an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO:11).

[0119] The specific amino acid sequence of the modified sericin 1 protein is not particularly limited, as it may vary depending on the type of Lepidoptera insect and the specific deletion mode. Examples include the amino acid sequence shown in SEQ ID NO: 12 or 13, an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 12 or 13, and an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 12 or 13.

[0120] (2) Modified sericin 3 protein The modified sericin 3 protein is a sericin 3 protein in which one or more repeat units are deleted in at least one of the two repeat sequence regions present in the amino acid region encoded by exon 3. For example, a wild-type silkworm sericin 3 protein includes the amino acid sequence shown in SEQ ID NO: 19.

[0121] The specific amino acid sequences of the two repeat sequence regions present in the amino acid region encoded by the third exon are not particularly limited, but the first repeat sequence region closer to the N-terminus contains a repeat unit consisting of 86 amino acids, and the second repeat sequence region closer to the C-terminus contains a repeat unit consisting of 8 amino acids.

[0122] Examples of the amino acid sequence of the first repeat sequence region include the amino acid sequence shown in SEQ ID NO:20; an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO:20; or an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO:20.

[0123] The amino acid sequence of the repeat unit in the first repeat sequence region is not particularly limited as long as it is an amino acid sequence consisting of 86 amino acids and is a sequence that is repeated with a certain degree of sequence identity. For example, the amino acid sequence may be an amino acid sequence consisting of the consensus sequence shown in SEQ ID NO: 57; an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 57; or an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 57. More specifically, the amino acid sequence of the repeat unit may be an amino acid sequence shown in any one of SEQ ID NOs: 58 to 69.

[0124] Examples of the amino acid sequence of the second repeat sequence region include the amino acid sequence shown in SEQ ID NO:21; an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO:21; or an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO:21.

[0125] The amino acid sequence of the repeat unit in the second repeat sequence region is not particularly limited as long as it is an amino acid sequence consisting of 8 amino acids and is a sequence that is repeated with a certain degree of sequence identity, for example, an amino acid sequence consisting of a consensus sequence selected from the group consisting of SEQ ID NOs: 67 to 70; or an amino acid sequence in which one or several amino acids have been added, deleted, and / or substituted in an amino acid sequence selected from the group consisting of SEQ ID NOs: 67 to 70.

[0126] The number of repeat units to be deleted is not particularly limited as long as the number of complete repeat units is reduced. The first repeat sequence region usually has 9 to 10 repeat units. On the other hand, the second repeat sequence region usually has 18 repeat units. Specific numbers of repeat units to be deleted in the modified sericin 3 protein include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 16 or more, 17 or more, 18 or more, 20 or more, 23 or more, 25 or more, 26 or more, 27 or more, and 28. For example, 28 or less, 27 or less, 26 or less, 25 or less, 23 or less, 20 or less, 18 or less, 15 or less, 10 or less, or 9 or less repeat units can be deleted in the modified sericin 3 protein.

[0127] Alternatively, for example, as described above in the modified sericin 1 protein, the number of complete repeat units may be reduced by deleting a portion of the repeat units. The number of complete repeat units to be reduced in this case is not particularly limited, and specifically, for example, the number of complete repeat units to be deleted may be reduced by the number exemplified as the number of repeat units to be deleted.

[0128] The specific deletion mode of the entire modified sericin 3 protein is not particularly limited. For example, the modified sericin 3 protein may be a modified sericin 3 protein lacking the entire first repeat sequence region (e.g., an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 20) and / or the entire second repeat sequence region (e.g., an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 21). Further, the modified sericin 3 protein may be a modified sericin 3 protein lacking either the entire first repeat sequence region (e.g., an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 20) or the entire third repeat sequence region (e.g., an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 21).

[0129] The specific amino acid sequence of the modified sericin 3 protein is not particularly limited, since it may vary depending on the type of Lepidoptera insect and the specific deletion mode. Examples include the amino acid sequence shown in SEQ ID NO: 22 or 23, an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 22 or 23, and an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 22 or 23.

[0130] (3) Peptide of interest The silk thread of the present invention may additionally contain a peptide of interest encoded by an exogenous gene. The type of peptide of interest may be appropriately selected depending on the purpose and is not particularly limited. Examples of the peptide of interest include peptide hormones such as insulin, calcitonin, parathormone, and growth hormone, cytokines such as epidermal growth factor (EGF), fibroblast growth factor (FGF), interleukin (IL), interferon (IFN), tumor necrosis factor α (TNF-α), and transforming growth factor β (TGF-β), other functional proteins such as immunoglobulins, serum albumin, enzymes, collagen, and fluorescent proteins, or fragments thereof (including chimeric peptides).

[0131] For example, fluorescent proteins include GFP, DsRed, TurboRFP and variants thereof, as well as various fluorescent proteins having different origins, such as Azami Green.

[0132] The number and types of peptides of interest are not particularly limited. The silk thread used in the present invention may contain, for example, one or more peptides of one type of interest, or multiple peptides of multiple types of interest. When multiple peptides of interest are contained, they may be contained in the form of a fusion protein, in which case the arrangement of each peptide is not particularly limited. For example, one or more peptides of interest may be introduced linked to each other at the same position, or distributed at different positions. If necessary, a tag sequence that can be used for detection or purification may be included.

[0133] The peptide of interest used in the method of the invention may be a protein or part thereof that is susceptible to thermal denaturation and / or to denaturation in basic conditions.

[0134] The origin of the peptide of interest is not particularly limited, and may be derived, for example, from an endogenous gene or an exogenous gene of the lepidopteran insect.

[0135] The method for introducing the gene is not particularly limited, and the gene can be introduced using gene recombination techniques known in the art.

[0136] The position at which the target peptide is contained in the silk thread of this embodiment is not particularly limited. For example, the target peptide may be contained in the fibroin fiber or in the paste-like component.

[0137] When the silk thread contains a peptide of interest in the fibroin fiber, the specific embodiment is not particularly limited. For example, the peptide of interest is directly or indirectly bound to a fibroin protein (including, for example, an L chain and / or an H chain). The peptide of interest can be introduced into one or more sites selected from the group consisting of the N-terminus, C-terminus or a region near there, or an internal region of the fibroin protein.

[0138] When the silk thread contains the peptide of interest in the paste-like component, the specific form is not particularly limited. For example, the peptide may be contained as a fusion protein with sericin, as a molecule separate from sericin, or both.

[0139] 2-3. Uses of silk thread Any product can be manufactured from the silk thread (raw silk and / or degummed silk) of this embodiment, specifically, for example, nonwoven fabrics, fillers, surgical sutures, woven fabrics, knitted fabrics, musical instrument strings, etc.

[0140] The term "nonwoven fabric" refers to a fiber sheet, web or pad in which the fibers are oriented in one direction or randomly and are bonded to each other by entanglement, fusion or adhesion, other than paper, woven fabric, knitted fabric, tufted fabric and felted felt.

[0141] As used herein, the term "woven fabric" refers to a flat (sheet) structure formed by intersecting warp and weft threads. The term "woven fabric" as used herein includes fabrics produced by any weaving method.

[0142] As used herein, the term "filler" broadly refers to any agent or material for filling any space. The term "filler" as used herein includes those classified as "filling materials."

[0143] In this case, any other fiber material may be included. Specific examples of the fiber material include, but are not limited to, natural fibers including other animal fibers such as wool and plant fibers such as cotton, semi-synthetic fibers such as acetate, regenerated fibers such as rayon, synthetic fibers such as polyester, and inorganic fibers such as glass fibers.

[0144] Products manufactured from the silk thread of this embodiment can be used for any purpose. For example, even if the silk thread contains a target peptide that is easily heat-denatured, raw silk and / or degummed silk that retains its activity can be obtained. In that case, the obtained silk thread can be used for any purpose in which the target peptide can exert its function.

[0145] 2-4.Effects The silk thread of the present invention has improved solubility in water of the paste-like component sericin compared to silk threads from wild-type lepidopteran insects, making it possible to relax the conditions for boiling cocoons and / or scouring compared to conventional conditions, thereby reducing the cost required for heating, the burden on the silk thread, and the burden on the environment, and also reducing the effort required for silk reeling.

[0146] In particular, the silk thread of the present invention can be provided with low temperatures, alkalinity, and surfactant usage during the refining process in which the degummed thread is separated and refined from the silk thread, thereby reducing the amount of heat, heating time, and wastewater treatment volume of the alkaline solution used in the refining process, and thus reducing the labor required for the refining process.

[0147] When the silk thread of the present invention contains a peptide of interest that may be inactivated by heat or basic conditions, the peptide of interest and / or the degummed silk thread can be separated and purified under milder degumming conditions than conventional methods without inactivating the function of the peptide of interest. In this case, for example, when the peptide of interest is linked to a fibroin protein, silk thread having the function of the peptide of interest can be separated and purified. In addition, for example, when the peptide of interest is not linked to a fibroin protein (for example, contained in a paste-like component), the peptide of interest can be eluted, separated and purified, and degummed thread not containing the peptide of interest can be separated and purified.

[0148] 3. Raw Silk Manufacturing Method 3-1. Overview A third aspect of the present invention is a method for producing raw silk. According to the method of this aspect, raw silk can be prepared at a lower temperature or in a shorter time.

[0149] 3-2.Process The method of this embodiment includes the essential steps of a cocoon spinning step, a contact step, a stringing step, and a reeling step, and also includes the optional steps of a rearing step, a cocoon harvesting step, a drying step, and a washing step. Each step will be described in detail below.

[0150] 3-2-1. Rearing process The "rearing step" is an optional step, which is a step of rearing the lepidopteran insect of the first embodiment. The method of rearing the lepidopteran insect is not particularly limited and any rearing technique known in the art can be used. For example, when the lepidopteran insect is a silkworm, it is recommended to refer to "General Theory of Silkworms; written by Takami Tsuyoshi, published by the National Silkworm Association" for rearing.

[0151] The feed used is not particularly limited. For example, natural feed such as natural leaves may be used, or artificial feed may be used. As feed for silkworms, for example, natural leaves of food tree species such as leaves of the genus Morus may be used, or artificial feed such as Silkmate L4M or original silkworm species for 1-3 instars (Nihon Nosan Kogyo) may be used. Artificial feed is preferred because it suppresses the occurrence of diseases, allows feeding of stable quality and amount, and can be reared in a sterile environment as necessary. An example of a simple method for rearing silkworms will be given below.

[0152] Sweeping is performed using eggs laid by an appropriate number (for example, 4 to 10) of females of the same species of lepidopteran insect (here, silkworms). The hatched larvae are transferred from the egg carrier to a container lined with dry-proof paper (paraffin-treated paper) that serves as a silkworm seat, and are fed with artificial feed such as Silkmate arranged on the dry-proof paper. In principle, the feed is replaced once for the 1st and 2nd instars, and once to three times for the 3rd instar. If there is a lot of leftover old feed, it is removed to prevent decay. For rearing 4th to 5th instar adult silkworm larvae, they are transferred to a large container and the number of larvae per container is adjusted appropriately. Depending on the humidity and conditions inside the container, the container may be covered with dry-proof paper, acrylic, or mesh lids. The rearing temperature is 25 to 28°C throughout all instars.

[0153] 3-2-2. Cocooning process The "cocoon-spinning step" is a step of making the lepidopteran insect of the first embodiment spin a cocoon. When a rearing step is carried out, the cocoon-spinning step can be carried out simultaneously with or after the rearing step.

[0154] "Cocooning" refers to the process by which a final stage lepidopteran insect forms a cocoon for pupation.

[0155] The method used in this step is not particularly limited. Any cocoon spinning method known in the art can be used. For example, in the case of silkworms, this step can be achieved by collecting mature silkworms on the 6th to 8th day of their final instar and transferring them to a cocoon. "Transferring" refers to transferring silkworms to a cocoon. Cocoon spinning can be carried out at, for example, 25 to 28°C, but the temperature can be appropriately selected depending on the type of lepidopteran insect, etc. The lepidopteran insect of the first embodiment then forms a cocoon within the brood.

[0156] The time for this step is not particularly limited as long as it is sufficient for the formation of cocoons. For example, it can be performed for 12 hours or more, 16 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 4 days or more, 5 days or more, or 6 days or more. The upper limit of the time is not particularly limited as long as the cocoons can be recovered. For example, it can be 30 days or less, 25 days or less, 21 days or less, 20 days or less, 15 days or less, 14 days or less, 13 days or less, 10 days or less, 7 days or less, 5 days or less, 4 days or less, 3 days or less, etc.

[0157] 3-2-3. Cocooning process The "cocoon harvesting process" is an optional process in which the cocoons are scraped off from the cocoons after the spinning process and collected. In this process, the fluff adhering to the periphery of the cocoons may be removed additionally.

[0158] The method used in this step is not particularly limited. Any method known in the art can be used. For example, the step may be performed manually or automatically using a cocoon harvesting device. As the cocoon harvesting device, a fluff remover that removes only fluff, or a fully automatic cocoon harvesting fluff remover that scrapes the cocoons from the cocoons and removes the fluff, etc., can be used.

[0159] 3-2-4. Drying process The "drying step" is an optional step in which the cocoons and / or the pupae within the cocoon cavity are dried to prepare dried cocoons.

[0160] As used herein, the term "cocoon cavity" refers to the space within a cocoon, i.e., the pupal chamber. For example, in the case of a silkworm cocoon, it refers to the space in which the silkworm larva, pupa, or adult is housed.

[0161] This step is usually carried out for the purpose of killing pupae and the like in the cocoon cavity and / or for long-term storage. Therefore, the necessity of this step can be determined taking into consideration the conditions such as the cocoons to be used and the necessity of storage. For example, when using cocoons that do not contain pupae in the cocoon cavity or when not storing the cocoons, this step does not need to be carried out.

[0162] The drying method used in this step is not particularly limited, and may be, for example, a method used for drying raw silkworm cocoons. Specific examples include air heat drying such as hot air drying, natural drying such as drying in the sun, drying by infrared rays, reduced pressure drying in which degassing and evaporation are performed using a vacuum pump or the like in a container, freeze drying in which water is evaporated while still frozen, air drying using hot or cold air, dehumidification drying using a dehumidifier or the like, drying by electromagnetic waves, or a combination thereof. Any of the methods may be performed manually or automatically using a machine or the like.

[0163] The cocoons may be subjected to additional treatment during drying. For example, when natural drying is performed, the cocoons may be frozen or refrigerated before or after the natural drying.

[0164] 3-2-5. Contact process The "contact step" is an essential step in which the cocoons are brought into contact with water or an aqueous solution at 4°C to 70°C. This step loosens the cocoon shell and prepares the cocoons to a state where they can be reeled. If a drying step is performed, this step can be performed after the drying step.

[0165] Here, the term "cocoon layer" refers to the wall of a cocoon that is made of silk threads. The method used in this step is not particularly limited. For example, any method that allows a solid (cocoons) and a liquid (water or an aqueous solution) to directly contact each other can be used. Specifically, for example, the cocoons can be contacted by immersing them in an organic solvent or the like, spraying, spraying or applying the organic solvent to the cocoons, or a combination thereof.

[0166] The contact can be performed by a known method used in boiling or scouring silkworm cocoons. Specific methods include a manual method, for example, a method in which the cocoons are hung from something or placed in a bag or the like and immersed in the liquid, or a method in which the liquid is sprayed using a machine. The hanging method, the bagging method, and the spraying method can be performed in accordance with the methods used in scouring, such as the hanging method, the bagging method, and the spraying method. The "hanging method" is a method in which the fabric or silk thread is hung from a pole or rod placed above or in a pot and immersed in the liquid in the pot, and is divided into the pole method, the rod method, the stick method, etc. depending on the method used. The "bag method" is a method in which the cocoons or silk thread are placed in a bag such as a cotton bag and immersed in the liquid. The "spraying method" is a method in which the liquid is sprayed on the silk thread by a machine.

[0167] When a method using immersion is used, it is not necessary for the entire cocoon to be under the liquid surface at once, but it is sufficient that the entire cocoon is eventually brought into contact with the water or aqueous solution by, for example, changing its position, mixing, rotating, etc. Even when a method other than immersion is used, it is preferable to carry out an operation such that the entire cocoon can be brought into contact with the water or aqueous solution by changing its position or rotating, etc.

[0168] The contact time is not particularly limited as long as the water or aqueous solution can penetrate the cocoon layer. For example, it can be determined in relation to the temperature and properties of the water or aqueous solution. In general, the lower the temperature, the longer the time, and the higher the temperature, the shorter the time. For example, it can be performed for 30 seconds or more, 1 minute or more, 1 minute 30 seconds or more, 2 minutes or more, 3 minutes or more, 3 minutes 30 seconds or more, 4 minutes or more, 4 minutes 30 seconds or more, 5 minutes or more, 5 minutes 30 seconds or more, 6 minutes or more, 8 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 60 minutes or more, 90 minutes or more, 2 hours or more, 3 hours or more, 6 hours or more, 8 hours or more, 12 hours or more, or 16 hours or more. The upper limit is not particularly limited, but can be, for example, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, 1 day or less, 20 hours or less, 18 hours or less, 16 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 3 hours or less, 2 hours or less, etc.

[0169] The contact can be carried out multiple times. In this case, the composition of the water or aqueous solution, the contact time, the temperature, etc. may be changed for each contact, or the water or aqueous solution used in the previous contact may be used as is.

[0170] The type of water or aqueous solution used in this step is not particularly limited. For example, water for boiling silkworm cocoons or a scouring agent used for scouring raw silk may be used. According to the method of this embodiment, even when an aqueous solution is used, it is not necessary to use a high pH or high concentration basic solution or a high concentration surfactant solution as has been conventionally used.

[0171] When an aqueous solution is used in this step, its composition is not particularly limited. Specific examples of the aqueous solution include aqueous solutions containing basic salts such as sodium carbonate, sodium hydrogen carbonate, and calcium carbonate, acids such as citric acid, acetic acid, and propionic acid, and surfactants such as nonionic surfactants, anionic surfactants, and cationic surfactants.

[0172] The concentration of the solute is not particularly limited. For example, it can be 0.01 (w / v)% to 20 (w / v), 0.05 (w / v)% to 15 (w / v), 0.1 (w / v)% to 10 (w / v), 0.3 (w / v)% to 7 (w / v), or 0.5 (w / v)% to 5 (w / v). Alternatively, it can be 0.01% owf to 20% owf, 0.05% owf to 15% owf, 0.1% owf to 10% owf, 0.3% owf to 7% owf, or 0.5% owf to 5% owf, based on the weight of the target silk thread. The concentration of the solute can be determined based on the type of solute and the desired pH.

[0173] For example, when a basic solution is used, the initial pH at the start of this process is not particularly limited, but can be, for example, 7 to 14, 7 to 11, 7 to 9, 7 to 8, etc. Furthermore, when an acidic solution is used, the initial pH at the start of this process is not particularly limited. For example, it can be 4 to 7, 5 to 7, 6 to 7, 6.5 to 7, etc. The pH may be determined based on an actual measurement, or based on a pH calculated from the degree of ionization or concentration. For measuring the pH, a known method using, for example, a pH test paper or a pH meter can be used. The concentration and pH need not be kept constant as long as they are generally within the above range during this process. Furthermore, for example, a solute and / or water may be added during this process, or the solution may be renewed.

[0174] The aqueous solution used in this step may further contain any solute as necessary, such as metal ions, buffers, pH adjusters, surfactants, and chelating agents.

[0175] The temperature of the water and aqueous solution used in this step is not particularly limited as long as it is 4°C to 70°C. For example, cold water (less than 15°C), room temperature water (15°C or more and less than 40°C), or warm water (40°C or more and less than 70°C) can be used. Specific lower limits of the temperature include, for example, 4°C, 5°C, 10°C, 15°C, and 20°C. Specific upper limits of the temperature include, for example, 70°C, 65°C, 60°C, 55°C, 50°C, 45°C, 40°C, 37°C, 35°C, 30°C, 25°C, 20°C, 15°C, and 10°C.

[0176] The amount of water or aqueous solution is not particularly limited. For example, when the contact is performed by immersion, the bath ratio, which indicates the total volume of the liquid when the weight of the silk thread is taken as 1, is usually 1:10 to 1:100 or 1:20 to 1:80. Specifically, this step can be carried out with a bath ratio of, for example, 1:0.5 or more, 1:1 or more, 1:1.5 or more, 1:2 or more, 1:5 or more, or 1:10 or more.

[0177] The water or aqueous solution used in this step may be prepared before this step or simultaneously with this step. Specifically, for example, ingredients may be added and / or renewed during this step. This step may be carried out using a known machine such as a cocoon cooker.

[0178] 3-2-6. Cleaning process The "washing step" is an optional step in which the silk threads are contacted with water or an aqueous solution to wash the cocoons. This step can be carried out after the contacting step. In particular, when the method includes multiple steps, this step can be carried out between each of the steps. For example, when a drying step is carried out, this step can be carried out between the drying step and the contacting step and / or after the contacting step, and when the contacting step is carried out multiple times, this step can be carried out between each contact.

[0179] The washing liquid used in this step is not particularly limited, but examples thereof include surfactants, water-soluble or volatile organic solvents such as toluene, benzene, and ethanol, and water.

[0180] This process can be carried out multiple times, in which case the composition and temperature of the liquid used may be the same each time or may be different each time.

[0181] 3-2-7. Cording process The "stringing step" is an essential step in which stringing is performed on cocoons that have been through the contact step.

[0182] "Reeling" refers to the process of searching for and pulling out the ends of the threads from the cocoons. This is usually done with a reeling broom or the like, but in this specification, reeling includes any process in which the ends of the threads are pulled out from the reeling cocoons, regardless of the means. As used herein, the term "thread end" refers to the end of a silk thread that has been released from a cocoon.

[0183] The method and conditions used in this step are not particularly limited. For example, a method normally used for stringing silkworm cocoons can be used. For example, this may be done by rubbing the surface of the cocoon in the water in which the cocoon is soaked during reeling (hereinafter often referred to as "reeling water" in this specification) with a stringing broom or the like. As long as a large amount of water is not lost within the cocoon cavity, it is not necessarily necessary to carry out this step with the cocoon soaked in water. In addition, the method for rubbing the cocoon surface is not particularly limited as long as it allows the thread to be pulled out.

[0184] The temperature and composition of the reeling water are not particularly limited, but for example, the reeling water used in normal reeling of silkworm cocoons may be used, or one exemplified in the contact step or washing step may be used. For example, when the present method is carried out successively with the contact step or washing step, the water or solution used in the contact step or washing step may be used as the reeling water as is, or may be exchanged for water or an aqueous solution of a different composition for the present step. Also, for example, after use in the present step, the water or solution may be further exchanged for water or an aqueous solution of a different composition for the subsequent reeling step.

[0185] 3-2-8. Reeling process The "thread reeling process" is an essential process in which thread is reeled from the thread obtained in the stringing process. This process can be carried out simultaneously with or after the stringing process.

[0186] Any one of the threads drawn out in the stringing process can be selected for reeling. For example, similar to the stringing process in the normal silkworm cocoon reeling method, the threads may be further drawn out until only one thread remains.

[0187] The method used in this step is not particularly limited. For example, a method commonly used in reeling silkworm cocoons can be used. In addition, since the main purpose of this step is to produce raw silk from the cocoons, any method that can reel silk from the cocoons can be used in this step.

[0188] This process may be carried out manually by hand reeling or counter reeling, or may be carried out by controlling the fineness using a counter reel or multi-row reeling machine for fixed size reeling, or an automatic reeling machine for fixed fineness reeling.

[0189] If necessary, it is possible to address defects in the yarn such as a tie, or to control the fineness of the yarn by doubling and / or twisting, etc. These methods are known in the art and are not particularly limited.

[0190] The method may involve carrying out a series of steps using an automatic silk-reeling machine capable of continuously reeling and stringing.

[0191] 3-3.Effects According to the method of this embodiment, raw silk can be produced under mild conditions such as low temperature and low basicity. Therefore, even when a target peptide that is easily denatured by heat and / or easily denatured under basic conditions is contained, raw silk can be obtained while retaining its activity. Furthermore, according to the method of this embodiment, heat treatment and harmful waste liquid are unnecessary or can be reduced, so raw silk can be produced under conditions with low environmental impact. Therefore, the method of the present invention can be used for various purposes, for example, to reduce the impact on silk threads or to reduce silk production costs.

[0192] 4. Manufacturing method of varnishing yarn 4-1. Overview The fourth aspect of the present invention is a method for producing a rotting yarn. The method of this aspect includes a scouring step as an essential step and a washing step as an optional step. According to the method of this aspect, a rotting yarn can be produced while suppressing protein denaturation.

[0193] 4-2.Process The method of this embodiment includes a scouring step as an essential step and a washing step as an optional step.

[0194] 4-2-1. Scouring process The "degumming step" is an essential step, in which the silk thread according to the second embodiment is degummed by contacting it with water or an aqueous solution at 4°C to 70°C.

[0195] The silk thread used in this step is not particularly limited as long as it is the silk thread described in the second embodiment. For example, it may be raw silk reeled by the method described in the third embodiment, or it may be silk thread in the form of a lump of silk thread that has not been reeled. When raw silk reeled by the method described in the third embodiment is used in this step, it is possible to produce a degummed silk from lepidopteran insects by carrying out the method described in this embodiment together with the method described in the third embodiment.

[0196] The silk used in the present method may be derived from a single individual lepidopteran insect, or may be a mixture derived from multiple individuals or species.

[0197] According to the method of this embodiment, refining can be performed without using enzymes or high temperature conditions, so the silk thread used in the method of this embodiment may be silk thread that contains proteins or parts thereof that are easily denatured by heat and / or that are easily decomposed by the enzymes used in enzymatic refining (e.g., papain, alcalase, ceryase, etc.).

[0198] In this step, water or an aqueous solution at 4° C. to 70° C. can be used. The water or aqueous solution used in this case can be the same as that described in the contact step of the third embodiment.

[0199] When the method of this embodiment is carried out in combination with the method of the third embodiment, the water or aqueous solution used in the contact step may be used in this step as is, or may be replaced with fresh water or aqueous solution and then used in this step.

[0200] The treatment time in this step is not particularly limited, but it can usually be longer than the time used in the contact step of the third embodiment.

[0201] The treatment applied to the silk thread prior to the present method is not particularly limited. For example, it may include a treatment at a high temperature (e.g., 80°C or higher) such as boiling cocoons, which involves normal heating, or all the treatments may be performed at a low temperature (e.g., a temperature below 80°C, such as 70°C or lower). By performing all the treatments at a low temperature, silk can be produced without applying a load to the silk thread that would denature the proteins.

[0202] 4-2-2. Cleaning process The "washing step" is an optional step in which the silk thread is washed by contacting the silk thread with water or warm water, etc. This step can be carried out simultaneously with or after the scouring step. This step may be carried out in accordance with the washing step of the third embodiment.

[0203] Effects The refining method of this embodiment removes sericin from silk thread. Furthermore, the degree of refining can be adjusted depending on the use of the silk thread after refining.

[0204] According to the refining method of the present invention, refining can be performed at a relatively low temperature and without using enzymes. Therefore, the method of the present invention can be used for various purposes, such as reducing the load on silk threads or reducing silk refining costs. In particular, it is possible to obtain a degummed thread and / or a silk lump that retains the activity of a target peptide that is easily denatured by heat and / or decomposed by the enzyme used in enzymatic refining.

[0205] 5. Method for producing the desired peptide 5-1. Overview A fifth aspect of the present invention is a method for producing a peptide of interest. According to the production method of this aspect, a peptide of interest can be efficiently produced from larvae of a lepidopteran insect.

[0206] 5-2.Materials In the method for producing a peptide of interest according to this embodiment, a lepidopteran insect is used as a peptide production system. The lepidopteran insect used in this embodiment is the lepidopteran insect according to the first embodiment having an exogenous gene.

[0207] The exogenous gene carried by the lepidopteran insect used in this embodiment is a gene encoding the target peptide to be produced by the method of this embodiment.

[0208] 5-3. Manufacturing method The production method of the present invention includes a recovery step as an essential step and a rearing step as an optional step. Each step will be described below.

[0209] 5-3-1. Rearing process The "rearing step" is an optional step of rearing the lepidopteran insect of the first embodiment that expresses the peptide of interest. This step may be performed when the method of this embodiment is performed starting from rearing the lepidopteran insect, but does not need to be performed when the peptide of interest is recovered from an already obtained silk thread according to the second embodiment. This step may be carried out in accordance with the rearing step of the third embodiment.

[0210] 5-3-2. Recovery process The "recovery step" is an essential step in which the target peptide accumulated in the lumen of the silk gland from the larvae of a lepidopteran insect is recovered.

[0211] In the lepidopteran insects used in this embodiment, the exogenous gene is expressed mainly from the late final instar of the larva, and the target peptide encoded by the gene is accumulated in the silk gland cells.

[0212] The method for recovering the target peptide from this lepidopteran insect is not particularly limited. For example, the target peptide may be directly recovered from the lumen of the silk gland (e.g., the middle or anterior silk gland), or may be recovered from silk secreted from the silk gland.

[0213] <Direct recovery from the silk gland lumen> The method of directly recovering the peptide from the lumen of the silk gland is not particularly limited. For example, a method of extracting the silk gland (e.g., the middle silk gland or the anterior silk gland) from the insect body at the late final instar to prepupal stage and directly recovering the peptide of interest can be used. As a specific method, any method known in the art can be used. For example, the silk gland can be extracted by anesthetizing a lepidopteran insect at the late final instar just before spinning on ice, incising the dorsal side, and extracting it with tweezers or the like (see Mori Yasushi, ed., New Biological Experiments with Silkworms, Sanseido, 1970, pp. 249-255). The extracted silk gland can be gently shaken in the extraction buffer at, for example, 0 to 10°C, preferably 0 to 5°C, to elute the peptide of interest into the buffer. If the peptide of interest is not a heat-sensitive peptide, the extraction can be performed at a temperature of 10 to 40°C. Then, impurities such as tissue fragments are removed by centrifugation or filtration, and the supernatant containing the peptide of interest can be collected.

[0214] Since the lepidopteran insect of the present invention has sericin with improved water solubility, the peptide of interest directly recovered from the silk gland can be efficiently eluted even at low temperatures. Furthermore, for example, when the peptide of interest is a peptide or protein with relatively low water solubility, it may be easier to separate the peptide from sericin with improved water solubility than before.

[0215] <Recovery from silk thread secreted from silk glands> In this case, a cocoon spinning step, a cocoon harvesting step, and / or a drying step can usually be performed. The specific contents of these steps can be performed in accordance with the "cocoon spinning step", "cocoon harvesting step", and "drying step" described in the third embodiment, respectively, and detailed explanations thereof will be omitted here.

[0216] The recovery method in this case can be carried out in accordance with the refining step described in the fourth embodiment, so only the different points will be described here.

[0217] In the method of this embodiment, it is preferable to select conditions that do not denature the target peptide. According to the method of this embodiment, for example, the target peptide can be eluted and collected even if the silk thread is immersed in water at 4°C. Therefore, in this embodiment, any conditions suitable for the target peptide can be selected and used without considering the elution efficiency.

[0218] The conditions used may be changed depending on the type of the target peptide, and conditions under which many water-soluble proteins are not denatured may be used regardless of the type of the target peptide. Specific conditions are not particularly limited, and the conditions described in the contact step described in the third embodiment may be used. Preferably, this step is carried out at a temperature lower than that of hot water. Also, this step is preferably carried out using water or an aqueous solution with a pH of about 6 to 8.

[0219] Unlike the scouring process, in this process, the peptide of interest is eluted in water or an aqueous solution, and the water or aqueous solution is then recovered. The recovery method is not particularly limited. Any method that can be used to separate liquids and solids can be used. For example, the liquid containing the peptide of interest can be recovered by removing the silk threads using a centrifuge or a net.

[0220] If necessary, the peptide of interest can be further purified. The method used for purification is not particularly limited. Any method commonly used for purifying peptides or proteins can be used. Examples of such methods include gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, chromatography such as HPLC, ammonium sulfate fractionation, ultrafiltration, and immunoadsorption, or a combination of two or more of these.

[0221] Effects According to this method, target peptides that are easily denatured can be efficiently recovered from silk thread.

[0222] In particular, the silk thread of the present invention allows the use of lower temperatures and lower amounts of basic and surface-active solutes, thereby enabling the recovery of the target peptide under conditions that reduce the risk of denaturation of the target peptide.

[0223] The silk thread removed in this method can be used as normal degummed thread as it is, so that the desired peptide and degummed thread can be obtained simultaneously from the silk thread without waste. EXAMPLES

[0224] <Example 1. Creation of a line lacking a partial deletion of sericin> (the purpose) A silkworm strain lacking the repeat sequence region of sericin is produced. (Methods and Results) 1. Design of TALEN expression vector The TALEN expression vector was constructed with reference to Y. Takasu et al. (2013) "Efficient TALEN construction for Bombyx mori gene targeting", PLoS One, 8, e73458. TALEN was designed to recognize the base sequences shown in Table 1 below.

[0225] [Table 1]

[0226] 2. TALEN mRNA Delivery Using each TALEN expression vector as a template, TALEN mRNA for introduction was synthesized using mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Invitrogen). The synthesized TALEN mRNA was injected into silkworm fertilized eggs 4 to 8 hours after laying to introduce the TALEN. The silkworm strain used for introduction was w1-pnd. The combinations of TALEN mRNA introduced into each line are shown in Table 2 below.

[0227] [Table 2]

[0228] 3. Individual selection The adults of the injected generation were crossed with the parent strain to obtain the next generation (F1). From the resulting F1 individuals, individuals that had the expected partial deletion and produced sericin protein with the expected molecular weight were selected.

[0229] (1) Confirmation of partial deletion Selection based on the presence or absence of partial deletion was performed by PCR using a primer pair with the nucleotide sequences shown in Table 3. Genomic DNA prepared from the F1 individual using ISOTISSUE (Nippon Gene) was used as a template for PCR. A primer set that only amplifies the partial deletion type and a primer set that only amplifies the wild type (WT) were mixed to confirm the sericin 1 line.

[0230] [Table 3]

[0231] (2) Confirmation of the sequence of the partially deleted gene The nucleotide sequence of each of the resulting partially deleted genes was confirmed by sequencing the nucleotide sequence using a SeqStudio Genetic Analyzer (Thermo Fisher Scientific).

[0232] As a result, it was found that the base sequence of the modified sericin 1 gene (Ser1 ΔR1) lacking the 6th exon is the base sequence shown in SEQ ID NO: 6, the base sequence of the modified sericin 1 gene (Ser1 ΔR2) lacking the 8th exon is the base sequence shown in SEQ ID NO: 7, the base sequence of the modified sericin 3 gene (Ser3 ΔR1) lacking the first repeat sequence region is the base sequence shown in SEQ ID NO: 17, and the base sequence of the modified sericin 3 gene (Ser3 ΔR2) lacking the second repeat sequence region is the base sequence shown in SEQ ID NO: 18.

[0233] Furthermore, the amino acid sequences of the modified sericin 1 protein and the modified sericin 3 protein were identified from these base sequences. The modified sericin 1 protein produced by the Ser1 ΔR1 line was identified as having the amino acid sequence shown in SEQ ID NO: 12, which is composed of 722 amino acids, the modified sericin 1 protein produced by the Ser1 ΔR2 line was identified as having the amino acid sequence shown in SEQ ID NO: 13, which is composed of 2762 amino acids, the modified sericin 3 protein produced by the Ser3 ΔR1 line was identified as having the amino acid sequence shown in SEQ ID NO: 22, which is composed of 396 amino acids, and the modified sericin 3 protein produced by the Ser3 ΔR2 line was identified as having the amino acid sequence shown in SEQ ID NO: 23, which is composed of 1085 amino acids.

[0234] (3) Expression of truncated sericin protein The expression of the partially deleted sericin protein as designed was confirmed by SDS-PAGE. Sericin was eluted by soaking approximately 7.5 mg of small pieces cut from the cocoons of the partially deleted and wild-type strains in 1 mL of 8 M urea / 2% SDS solution and incubating at 80°C for 30 minutes. SDS-PAGE was performed using 4-12% NuPage Bis-Tris Gel (Thermo Fisher Scientific) according to the manufacturer's protocol based on the obtained sericin solution. After electrophoresis, the gel was stained with CBB (Bio-Rad) or Oriole (Bio-Rad) and observed.

[0235] 3. Establishment of homozygous strains The selected individuals were crossed with the white c strain, and then individuals with the deleted protein were crossed with each other to homozygize each strain. The homozygous strains were then examined for the presence or absence of the partial deletion and the size of the protein, in the same manner as in the selection.

[0236] The results obtained for each line are shown in Figures 9 to 12, B and C, respectively. As shown in each figure, B, in each line, individuals homozygous for the partial deletion as expected were obtained using the primers used (white arrows in each figure, A).

[0237] As shown in each figure C, in all strains, individuals were obtained that did not produce the target wild-type sericin ("Sericin 1C" for sericin 1 and "Sericin 3" for sericin 3), and only produced short modified sericins with the expected length.

[0238] <Example 2. Measurement of reduction rate> (the purpose) The efficiency of sericin elution was examined using silk obtained from a partially sericin-deleted line.

[0239] (method) Cocoon samples for measurement were approximately 7.5 mg pieces cut from cocoons of each of the sericin partial deletion strains and the wild-type strain (white c). The elution of sericin with water was carried out by incubating the cocoon samples in 1 mL of purified water at 4°C, 20°C, 40°C, 60°C or 80°C for 30 minutes. As a positive control, a cocoon sample incubated in 1 mL of 0.005% sodium carbonate solution at 100°C for 30 minutes was used.

[0240] The solution collected after incubation was subjected to SDS-PAGE, and the reduction rate was calculated from the cocoon sample. SDS-PAGE was performed in the same manner as in Example 1. The experiment was performed in triplicate.

[0241] The reduction rate was calculated using the following formula based on the dry weight of the cocoon samples before and after incubation. (Reduction rate (%)) = {(weight before incubation) - (weight after incubation)} / (weight before incubation) × 100

[0242] The extraction rate of sericin was calculated by comparing the reduction rate in the treatment with 0.005% sodium carbonate solution and the treatment with water using the following formula. (Dissolution (%)) = (Reduced rate in water) / (Reduced rate in sodium carbonate solution) x 100

[0243] (result) The results are shown in Figures 13 to 20. Figures 13 to 16 show the results of SDS-PAGE. Figures 17 and 18 show the results of reduction rate, and Figures 19 and 20 show the results of elution rate of sericin.

[0244] As previously known, in the wild type, no sericin was observed to be eluted when incubated in water at temperatures below 60°C, and a faint sericin band was detected only after incubation at 80°C (row "WT" in Figures 13 to 16). In contrast, in each deletion line, a strong sericin band was detected even in water under all temperature conditions confirmed. This confirmed that the partial deletion line of the present invention is capable of eluting sericin even when incubated in water at a relatively low temperature.

[0245] This result was also confirmed by the results of the reduction rate (Figures 17 and 18). In the wild type, the reduction rate was about 3% ("WT" in Figures 17 and 18). On the other hand, the reduction rate in the modified sericin line 1 of the present invention was about 10% even at 4°C (Figure 17), and the reduction rate in the modified sericin line 3 of the present invention was about 8% even at 4°C (Figure 18). Overall, there was a tendency for the reduction rate to increase slightly with temperature, but no significant difference was observed between low temperature conditions (4°C) and high temperature conditions (80°C). This demonstrated that the modified sericin line of the present invention allows for good refining even at lower temperatures than conventionally and without the use of solutes such as bases.

[0246] Furthermore, the results shown in Figures 13 to 16 were analyzed with respect to the elution rate of sericin (Figures 19 and 20). As with the results of the reduction rate, the elution rate of sericin in the wild type was low at about 10% ("WT" in Figures 19 and 20). On the other hand, the elution rate in the modified sericin 1 line of the present invention was about 35% even at 4°C (Figure 19), and the elution rate in the modified sericin 3 line of the present invention was about 30% even at 4°C (Figure 20). As with the reduction rate, the elution rate of sericin also tended to increase somewhat with temperature overall, but no significant difference was observed between low temperature conditions (4°C) and high temperature conditions (80°C). This demonstrated that the modified sericin line of the present invention can easily extract proteins such as sericin from the surface of silk thread even under more relaxed conditions than before.

[0247] Example 3: Measurement of extraction efficiency of target peptide (the purpose) The extraction efficiency of the target peptide expressed in silk obtained from a partially deleted line of sericin was examined.

[0248] (method) Each of the sericin partial deletion strains was crossed with a silkworm strain that produces cocoons containing EGFP (expressing EGFP in the posterior silk gland). Through multiple generations of crossing, heterozygous and homozygous individuals for the partial sericin deletion were obtained. Cocoon samples for measurement were approximately 7.5 mg pieces cut from the cocoons of each individual. The target peptide, EGFP, was extracted by incubating the cocoon samples in 1 mL of purified water overnight at 4°C. The EGFP concentration in the extract was measured by ELISA.

[0249] The relative extraction efficiency was calculated as the ratio to the EGFP concentration in the wild-type silkworm using the following formula. (Relative efficiency) = (EGFP concentration of each line) / (wild-type EGFP concentration)

[0250] (result) The results are shown in Table 4.

[0251] [Table 4]

[0252] As with the reduction rate, it was confirmed that 1.2 to 1.9 times more EGFP could be extracted from each deletion line compared to the wild-type EGFP-expressing line. As with the tendency observed with the reduction rate, it was also suggested that whether the genotype was homozygous or heterozygous did not have a significant effect on the elution efficiency.

[0253] This shows that by crossing the sericin partial deletion line of the present invention with an existing exogenous gene expression line, an exogenous gene expression line partially deficient in sericin can be obtained, and that this line makes it possible to extract the target peptide more efficiently than a line having only the complete sericin gene.

[0254] Example 4: Relationship between incubation time and reduction rate (the purpose) The relationship between the reduction rate and incubation time in silk obtained from a line lacking partial sericin was examined.

[0255] (method) The experiment was carried out in the same manner as in Example 2, except that small pieces cut from cocoons of heterozygous and homozygous individuals of each Ser1 exon 6 deletion line (Ser1 ΔR1) were used as cocoon samples for measurement and incubated in water at 4° C. for 2 minutes, 5 minutes, 10 minutes, 20 minutes, and 30 minutes. The experiment was carried out in triplicate.

[0256] (result) The results are shown in Figure 21. As shown in FIG. 21, the change in the reduction rate with incubation time showed a similar tendency regardless of whether homozygous or heterozygous individuals were used.

[0257] In both cases, even though 4°C water was used, a reduction rate of 10% or more was achieved when the material was incubated for at least 5 minutes, indicating that sufficient scouring was possible. With longer incubation times, no significant change in the reduction rate was observed.

[0258] This demonstrates that the sericin partial deletion line of the present invention not only enables degumming under low-stress conditions such as low-temperature water, but also enables degumming in an extremely short period of time.

Claims

1. A lepidopteran insect capable of producing a modified sericin 1 protein, wherein the gene encoding the modified sericin 1 protein includes all of exon 7 and is missing all or part of exon 6 and / or exon 8.

2. 2. The lepidopteran insect of claim 1, wherein the base sequence of the 7th exon has a base sequence that has 90% or more base identity with the base sequence shown in SEQ ID NO: 4, the base sequence of the 6th exon has a base sequence that has 90% or more base identity with the base sequence shown in SEQ ID NO: 3, and the base sequence of the 8th exon has a base sequence that has 90% or more base identity with the base sequence shown in SEQ ID NO:

5.

3. A lepidopteran insect capable of producing a modified sericin 3 protein, wherein the gene encoding the modified sericin 3 protein is missing all or part of a base sequence having 90% or more base identity with the base sequence shown in SEQ ID NO: 15 or a base sequence having 90% or more base identity with the base sequence shown in SEQ ID NO:

16.

4. The lepidopteran insect of claim 1 , further comprising an exogenous gene.

5. The lepidopteran insect of claim 4 , wherein the exogenous gene is linked to a promoter operable in the silk gland.

6. A method for producing raw silk, comprising the steps of: A cocoon spinning process for forming a cocoon by the lepidopteran insect according to any one of claims 1 to 5; A contacting step of contacting the cocoons with water or an aqueous solution at 4°C to 70°C; A stringing step of stringing the cocoons; a reeling process in which raw silk is obtained by reeling the thread obtained in the stringing process; Including, The manufacturing method.

7. 1. A method for producing a peptide of interest, comprising the steps of: The peptide of interest is a peptide encoded by an exogenous gene carried by a lepidopteran insect according to claim 4 or 5, A recovery step of recovering the target peptide accumulated in the lumen of the silk gland from the lepidopteran insect larvae. The manufacturing method.

8. A silk thread comprising a modified sericin protein, wherein one or more repeat units are deleted in a repeat sequence region of the modified sericin protein, The silk thread, wherein the modified sericin protein comprises a modified sericin 1 protein or a modified sericin 3 protein.

9. the modified sericin 1 protein comprises an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO:10, The repeat sequence region is at least one of an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 9 and an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO:

11. The silk thread according to claim 8.

10. 10. The silk thread of claim 9, wherein the repeating unit consists of the amino acid sequence shown in SEQ ID NO: 24 or 45.

11. The silk thread described in claim 9 or 10, wherein the modified sericin 1 protein is missing all of the amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 9, or all of the amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO:

11.

12. the modified sericin protein comprises a modified sericin 3 protein; The silk thread described in claim 8, wherein the repetitive sequence region is at least one of an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO:

21.

13. The silk thread according to claim 12, wherein the repeating unit consists of an amino acid sequence shown in SEQ ID NO: 57, and / or any one selected from the group consisting of SEQ ID NOs: 67 to 70.

14. The silk thread described in claim 12 or 13, wherein the modified sericin 3 protein lacks the entire amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 20 or the entire amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO:

21.

15. A method for producing varnishing, comprising the steps of: The method includes a refining step of contacting the silk thread according to claim 8 with water or an aqueous solution at 4 ° C to 70 ° C. The manufacturing method.

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