CN105683373A - Exogenous gene expression enhancer - Google Patents

Abstract

The subject of the present invention is to develop and provide simple and effective suppression of sericin and/or silk fibroin H chain gene expression as the main protein specific to silk glands, so as to efficiently produce target proteins using silk glands in genetically recombinant silkworms A method as a mass production system of proteins. The present invention provides a silkworm system capable of expressing antisense RNA in the silkworm. The antisense RNA uses the repeat sequence region of sericin and/or silk fibroin H chain as a target site.

Description

exogenous gene expression enhancer

technical field

The present invention relates to an exogenous gene expression enhancer which uses an antisense RNA expression vector as an active ingredient in a protein expression system utilizing the silk gland of silkworm, and a genetically recombined silkworm having the antisense RNA expression vector.

Background technique

The silk gland of the silkworm (Bombyxmori) has the ability to synthesize a large amount of protein in a short time. In addition, since the silk gland of the silkworm is a large organ, it is easy to extract, and the synthesized protein is stored in the lumen of the silk gland, so it also has the advantage of being easy to recycle. Therefore, the genetically recombined silkworm that expresses the target protein using the silk gland is considered promising as a mass production system for proteins with high added value such as useful proteins or high-performance silk.

Bombyx mori silk glands are morphologically a pair of left and right organs as shown in Figure 1, which are composed of three regions: anterior silk gland, middle silk gland and posterior silk gland. In the middle silk gland cells, water-soluble gelatin-like proteins, sericin 1 (in this specification, often abbreviated as "Ser1") and sericin 2 (in this specification, often abbreviated as "Ser2") and sericin 3 (in this specification, often abbreviated as "Ser3") (Fig. 1). These proteins are secreted into the lumen of the middle silk gland after synthesis. In addition, in the posterior silk gland cells, three main proteins constituting silk fibroin, which is a component of silk fibers, are synthesized, namely silk fibroin H chain (in this specification, it is often abbreviated as "FibH"), silk fibroin L chain (in this specification, it is often abbreviated as "FibL") and p25/FHX (hereinafter, referred to as "p25"). These three proteins form a complex (silk fibroinelementary unit; in this specification, hereinafter referred to as "SFEU complex") at a ratio of FibH:FibL:p25=6:6:1, and are secreted into the lumen of the posterior silk gland. Then, the SFEU complex is transferred to the lumen of the middle silk gland, covered with sericin, and spun from the front silk gland as silk ( FIG. 1 : Non-Patent Document 1). Therefore, when utilizing the silk gland of silkworm as a protein expression system, the gene expression system specifically expressed in the middle silk gland or the posterior silk gland may be used. In fact, as a recombinant protein expression system using the middle silk gland, a mass expression method using the GAL4/UAS system (Non-Patent Document 2) or a system combining the Ser1 promoter and the Hr3 enhancer (Non-Patent Document 2) has been established so far. Document 3).

In the protein expression system using the silk gland of silkworm, in order to improve the production efficiency of the target protein, the expression level of the gene itself is increased, and the expression level of the target protein is relatively increased by reducing the amount of other main proteins of the silk gland, and from the silkworm Efficient recovery of the target protein is important. One of the reasons for hindering protein recovery is the presence of intercalated proteins. For example, silk fibroin and sericin which are main proteins of silk glands are mentioned. Since these proteins are synthesized in a large amount and are of fibrous quality with high viscosity and crystallinity, they are likely to cause clogging of columns or filters during extraction or purification of the target protein. Therefore, for efficient protein recovery, gene expression of these entrapped proteins needs to be suppressed. On the other hand, since excessive expression suppression of these genes expressed in the silk gland of silkworm silkworm affects the growth itself of silkworm, a technique for controlling the expression suppression is required.

In the silk gland of silkworm, the method of suppressing the expression of endogenous silk fibroin or sericin includes a method of using an existing gene mutation system and a method of artificially suppressing the expression of these genes.

As an example of a method using an existing gene mutation system, it is known that the FibH chain expressed from the posterior silk gland in the silkworm has an abnormal Nd system, and the FibL chain has an abnormal Nd-s system and Nd-sD system. Since the expression of the FibH chain in the Nd system or the expression of the FibL chain in the Nd-s system and the Nd-sD system is very low, the expression level of the target protein can be increased by using these systems using the middle silk gland (Patent Document 1) . However, the above-mentioned gene mutation system cannot suppress gene expressions such as Ser1 expressed by the middle silk gland, and there is no report on the mutation system of genes expressed by the middle silk gland of the silkworm silkworm.

An example of a method for artificially suppressing gene expression includes the RNAi (RNA interference) method. The RNAi method is a method for suppressing (silencing) the expression of a target gene in vivo through degradation of the transcription product of the target gene. In general, the RNAi method uses siRNA (small interfering RNA) or miRNA (microRNA) composed of double-stranded RNA, and shRNA (shorthairpinRNA) composed of single-stranded RNA that forms a double-stranded RNA region within the molecule through self-folding. RNAi molecule. On the other hand, antisense RNA (in this specification, often referred to as "asRNA"), which is composed of single-stranded RNA and does not form a double-stranded RNA region in the molecule, anneals to the target mRNA, and finally exerts the effect of RNAi through the same process as double-stranded RNA. Inhibitory effect on gene expression. However, since its effect is very low compared with other RNAi molecules, it is hardly used (Non-Patent Document 4).

In insects of the order Lepidoptera including the silkworm, the effect of gene expression suppression by RNAi is generally low compared with other biological species (Non-Patent Document 5). However, its effect is known to vary depending on the tissue, and even in Lepidoptera, it is only relatively high in the skin or silk glands. For example, it has been reported that expression of each gene can be suppressed by expressing double-stranded RNA of FibH chain, FibL chain, and Ser1 in silkworm cells (Patent Documents 2 to 4). However, these methods have the problem of high production cost because the double-stranded RNA must be expressed and constructed in vivo, and the production of the expression vector or the production of the genetically modified silkworm is complicated and labor-intensive. Furthermore, in the prior art, there was a problem that it was very difficult to combine a plurality of genes and simultaneously suppress their expression. These problems can be overcome by asRNA composed of single-stranded RNA. However, it has been reported that the effect of suppressing gene expression by asRNA in the silk gland of silkworm silkworm is also very low, and is only about a few hundredths compared with double-stranded RNA (Non-Patent Document 6).

prior art literature

patent documents

Patent Document 1: Japanese Patent Laid-Open No. 2004-135528

Patent Document 2: Japanese Special Application Form 2006-521802

Patent Document 3: Japanese Patent Laid-Open No. 2008-245623

Patent Document 4: Japanese Patent Laid-Open No. 2011-103816

non-patent literature

Non-Patent Document 1: InoueS.etal., 2000, The Journal of Biological Chemistry, 275(51): 40517-40528.

Non-Patent Document 2: Tatematsu K.etal., 2010, Transgenic Research, 19(3):473-87.

Non-Patent Document 3: Tomita M. et al., 2007, Transgenic Research, 16(4):449-465.

Non-Patent Document 4: Tavernarakis N.etal., 2000, Naturegenetics, 24180-183.

Non-Patent Document 5: TereniusO.etal., 2011, Journal of Insect Physiology, (57): 231-245

Non-Patent Document 6: IsobeR.etal., 2002, Journalofinsectbiotechnologyandsericology, 71, 43-47

Contents of the invention

The object of the present invention is to develop and provide a method for effectively suppressing the gene expression of sericin and/or FibH chain, which is an endogenous main protein of silk gland and can become an intercalation protein, to recombine the silk gland of silkworm silkworm To improve the production efficiency of the target protein.

In order to solve the above-mentioned problems, the inventors of the present invention conducted intensive studies, and finally found that by expressing asRNA in the silk gland of silkworm silkworm, the repeat sequence region contained in the amino acid sequence of sericin and FibH chain as a direct target region, Even asRNA can effectively inhibit the gene expression of sericin and FibH chain. The present invention provides the following inventions based on the research results.

(1) An exogenous gene expression enhancer of silk gland of silkworm, which contains an asRNA expression vector capable of specifically expressing at least one asRNA selected from Ser1asRNA, Ser2asRNA and Ser3asRNA in silk gland of silkworm middle part and/or capable of making An asRNA expression vector specifically expressing Fib-HasRNA in the posterior silk gland of Bombyx mori is used as an active ingredient, wherein Ser1asRNA contains at least one repeat of the repeat sequence region at position 628 to position 1054 in the amino acid sequence of silkworm Ser1 represented by the coding sequence number 1 A sequence complementary to the base sequence of the unit, Ser2asRNA contains a base sequence complementary to the base sequence of at least one repeat unit in the repeat sequence region of positions 41 to 117 or positions 685 to 1359 in the amino acid sequence of Bombyx mori Ser2 represented by the coding sequence number 4 Sequence, Ser3asRNA contains the base sequence complementary to the base sequence of at least one repeating unit in the repeat sequence region of 141-978 or 1031-1178 in the amino acid sequence of Bombyx mori Ser3 represented by the coding sequence number 8, and Fib-HasRNA contains A sequence that is complementary to the base sequence of at least one repeat unit in the repeat sequence region at position 152 to position 5203 in the amino acid sequence of the silkworm FibH chain represented by sequence number 12.

(2) The exogenous gene expression enhancer according to (1), wherein the repeating unit of Ser1 is composed of the amino acid sequence represented by sequence number 2, the repeating unit of Ser2 is composed of the amino acid sequence represented by sequence number 5 or 6, and the repeating unit of Ser3 is composed of the amino acid sequence represented by sequence number 5 or 6. The repeating unit of the FibH chain consists of the amino acid sequence represented by sequence number 9 or 10, and the repeating unit of the FibH chain consists of the amino acid sequence represented by sequence number 13.

(3) The exogenous gene expression enhancer according to (1), wherein the asRNA of Ser1 is composed of the base sequence represented by SEQ ID NO: 3, the asRNA of Ser2 is composed of the base sequence represented by SEQ ID NO: 7, and the asRNA of Ser3 is composed of the base sequence represented by SEQ ID NO: 7. It consists of the base sequence represented by SEQ ID NO: 11, and the FibH chain asRNA consists of the base sequence represented by SEQ ID NO: 14.

(4) The exogenous gene expression enhancer according to any one of (1) to (3), wherein the asRNA expression vector is composed of two subunits.

(5) A genetically recombined silkworm, which has an asRNA expression vector capable of specifically expressing at least one asRNA selected from Ser1asRNA, Ser2asRNA and Ser3asRNA in the middle silk gland of silkworm and/or capable of specifically expressing Fib in the rear silk gland of silkworm The asRNA expression vector of -HasRNA, wherein, Ser1asRNA contains the sequence complementary to the base sequence of at least one repeating unit in the repeat sequence region of 628-1054 in the amino acid sequence of Bombyx mori Ser1 represented by the coding sequence number 1, and Ser2asRNA contains the sequence complementary to the coding A sequence complementary to the base sequence of at least one repeating unit in the repeat sequence region at positions 41 to 117 or 685 to 1359 in the amino acid sequence of Bombyx mori Ser2 represented by sequence number 4, and Ser3asRNA contains the Ser3 of silkworm represented by the coding sequence number 8 In the amino acid sequence of 141-978 or 1031-1178 in the repeated sequence region, the base sequence of at least one repeating unit is complementary, and Fib-HasRNA contains the amino acid sequence of the silkworm FibH chain represented by the coding sequence number 12 A sequence complementary to the base sequence of at least one repeat unit in the repeat sequence region from position 152 to position 5203.

(6) The exogenic recombinant silkworm according to (5), wherein the repeating unit of Ser1 is composed of the amino acid sequence represented by sequence number 2, the repeating unit of Ser2 is composed of the amino acid sequence represented by sequence number 5 or 6, and the repeating unit of Ser3 is composed of the amino acid sequence represented by sequence number 5 or 6. It consists of the amino acid sequence represented by SEQ ID NO: 9 or 10, and the repeating unit of the FibH chain consists of the amino acid sequence represented by SEQ ID NO: 13.

(7) The genetically recombined silkworm according to (5), wherein the asRNA of Ser1 consists of the base sequence represented by SEQ ID NO: 3, the asRNA of Ser2 consists of the base sequence represented by SEQ ID NO: 7, and the asRNA of Ser3 consists of the base sequence represented by SEQ ID NO: 11 The nucleotide sequence shown in , and the FibH chain asRNA is composed of the nucleotide sequence shown in SEQ ID NO: 14.

(8) The genetically recombinant silkworm according to any one of (5) to (7), wherein the expression vector consists of two subunits.

(9) The genetically recombinant silkworm according to (8), wherein the two subunits are present on different chromosomes.

(10) The genetically recombinant silkworm according to any one of (5) to (9), which has an exogenous gene expression vector capable of expressing a target exogenous gene.

(11) A method for producing a gene-recombined silkworm, comprising the step of administering the exogenous gene expression enhancer described in any one of (1) to (4) to the silkworm.

(12) A method for producing a target peptide encoded by an exogenous gene in the silk gland of the genetically recombinant silkworm described in (10).

This specification includes the contents described in the specification and/or drawings of Japanese Patent Application No. 2013-222138 on which the priority of this application is based.

According to the exogenous gene expression enhancer of the present invention, when administered to silkworms, it is possible to suppress the synthesis of sericin or FibH chains, which are inclusion proteins, in silkworms in the protein production system.

According to the genetically recombined silkworm of the present invention, the silkworm in which the synthesis of sericin and/or FibH chain is inhibited can be provided. Moreover, the production efficiency and recovery rate of the target peptide encoded by the exogenous gene can be improved if it is the gene recombinant silkworm which further has the exogenous gene expressed from the silk gland.

According to the peptide production method of the present invention, by using the genetically modified silkworm of the present invention as a protein production system, it is possible to produce a large amount of protein with high added value at relatively low cost.

Description of drawings

Fig. 1 shows a conceptual diagram before the silk fibroin constituting protein expressed in silk glands and various parts of the silkworm and silk silk is extruded.

Fig. 2 is a diagram showing a configuration example of the first subunit and the second subunit when the asRNA expression vector, which is the active ingredient of the exogenous gene expression enhancer of the present invention, consists of two gene expression units. A to C are configuration examples of the first subunit. A is a part of the posterior silk gland GAL4 expression vector which arrange|positioned GAL4 gene as the DNA which encodes a transcription regulator under the posterior silk gland promoter. B is a part of the middle silk gland GAL4 expression vector that configures the GAL4 gene under the middle silk gland promoter. C is a part of the middle & posterior silk gland GAL4 expression vector in which the GAL4 gene is arranged under the respective promoters of the middle silk gland and the posterior silk gland. D and E are configuration examples of the second subunit. D is a part of the asRNA expression vector having one asRNA-encoding DNA downstream of the UAS promoter, which is the target promoter of GAL4. E is part of an asRNA expression vector with asRNA-encoding DNA in 2 tandems downstream of the UAS promoter. The arrows of the 3'UTR indicated by white boxes in each subunit indicate the direction of transcription.

Fig. 3 is a diagram showing the structure of the second subunit expressing Ser1asRNA used in Example 1 and the like. A is the UAS-5'Ser1asRNA expression vector expressing 5'Ser1asRNA. In addition, B is a UAS-3'Ser1asRNA expression vector expressing 3'Ser1asRNA. Here, 5'Ser1asRNA targets the 5' region of Ser1 that does not contain a repeat sequence region. In addition, 3'Ser1asRNA targets the 3' region of Ser1 that contains the repeat region. The arrow of Ser1asRNA-encoding DNA indicates the direction of transcription (hereinafter, the same as this).

Fig. 4 is a diagram showing the structure of the second subunit expressing Ser1shRNA used in Example 1 and the like. A is the UAS-5'Ser1shRNA expression vector expressing 5'Ser1shRNA. In addition, B is a UAS-3'Ser1shRNA expression vector expressing 3'Ser1shRNA. Ser1 shRNA is composed of Ser1 sense RNA (Ser1sRNA), linker (actin intron) and Ser1asRNA.

It is a figure which shows the structure of the 1st subunit used in Example 1 etc., and shows the middle part silk gland GAL4 expression vector which expresses GAL4 gene in the middle part silk gland.

Fig. 6 is a graph showing the expression inhibition of the Ser1 gene of the genetically recombinant silkworm silkworm having each of the second subunits shown in Figs. 3 and 4 and the first subunit shown in Fig. 5 . The % described above in the figure is the average relative amount of the 3 systems (1, 2, 3) represented by each group.

Fig. 7 is a graph showing SDS-PAGE of the cocoon protein of the genetically recombinant silkworm having each of the second subunits shown in Figs. 3 and 4 and the first subunit shown in Fig. 5 .

Fig. 8 is a diagram showing the structure of the second subunit expressing Fib-HasRNA used in Example 2 and the like. A is the UAS-Fib-H1asRNA expression vector expressing Fib-H1asRNA. In addition, B is a UAS-Fib-H2asRNA expression vector expressing Fib-H2asRNA. Fib-H1 and Fib-H2 differ in the length of the repeat region of the target FibH chain.

It is a figure which shows the structure of the 1st subunit used in Example 2 etc., and shows the posterior silk gland GAL4 expression vector which expresses GAL4 gene in posterior silk gland.

Fig. 10 is a graph showing the suppression of expression of the FibH chain gene of the genetically recombinant silkworm silkworm having the second subunit shown in Fig. 8 and the first subunit shown in Fig. 9 .

Fig. 11 is a diagram showing SDS-PAGE of the silk gland protein of the genetically recombinant silkworm silkworm having the second subunit shown in Fig. 8 and the first subunit shown in Fig. 9 .

Fig. 12 is a diagram showing the configuration of the second subunit (A) and the first subunit (B) used in Example 3 and the like. The second subunit represented by A is a UAS-3'Ser1-FibHasRNA expression vector that can simultaneously express Ser1asRNA and FibHasRNA through the serial arrangement of Ser1asRNA-encoding DNA and FibHasRNA-encoding DNA downstream of the UAS promoter. In addition, in B, the GAL4 gene is placed downstream of the Ser promoter (Ser-pro) which is the middle silk gland promoter and the FibH promoter (Fib-Hpro) which is the rear silk gland promoter, and the middle and rear silk glands are used. A GAL4 expression vector for the middle & posterior silk glands that simultaneously expresses the GAL4 gene in the upper silk glands.

Figure 13 shows that there is the middle part & back silk gland GAL4 expression vector shown in Figure 12B as the 1st subunit, the middle part silk gland GAL4 expression vector shown in Figure 5 or the back part silk gland GAL4 expression vector shown in Figure 9 , and the SDS-PAGE figure of the silk gland protein of the genetically recombined silkworm silkworm of the UAS-3'Ser1-FibHasRNA expression vector shown in FIG. 12A as the second subunit.

Fig. 14 is a diagram showing the structure of an exogenous gene expression vector. A is the basic expression vector of the exogenous gene expression vector used in Examples 4 and 5. B is the UAS-EGFP expression vector used in the examples by replacing the marker gene of the expression vector of A with 3xP3-AmCyan from 3xP3-EGFP.

Fig. 15 is a graph showing the amount of EGFP protein per gene recombinant silkworm. The gene recombinant silkworm used here has the UAS-3'Ser1asRNA expression vector shown in Figure 3 or the UAS-3'Ser1shRNA expression vector shown in Figure 4 as the second subunit, and the middle part shown in Figure 5 as the first subunit. Silk gland GAL4 expression vector, and the UAS-EGFP expression vector shown in Figure 14 as an exogenous gene expression vector.

Fig. 16 is a graph showing the amount of EGFP protein per gene recombinant silkworm. The genetically recombined silkworm used here has the UAS-3'Ser1-Fib-HasRNA expression vector as the second subunit shown in Figure 12 and the middle & posterior silk gland GAL4 expression vector as the first subunit, and Figure 14 Indicates the UAS-EGFP expression vector.

Fig. 17 is a graph showing inhibition of gelation of silk gland extract by expression of Ser1asRNA and Fib-HasRNA.

detailed description

1. Exogenous gene expression enhancer

1－1. Summary

The first embodiment of the present invention is an exogenous gene expression enhancer. The exogenous gene expression enhancer of the present invention is characterized in that it contains, as an active ingredient, an expression vector of antisense RNA targeting the sericin gene or FibH chain gene expressed in the silk gland of silkworm silkworm. By administering the exogenous gene expression enhancer of this embodiment to silkworm, the gene expression of sericin and/or FibH chain can be suppressed in silk gland.

1－2. Definition

The main terms used in this specification are defined below.

"Exogenous gene" is a foreign gene introduced from the outside through artificial manipulation, and is a gene that can be expressed in the silk gland of the silkworm. The exogenous gene in this specification also includes a gene existing in a state inserted into a chromosome, such as a progeny of a recombinant, as long as its source is a gene introduced from the outside by human manipulation.

The "target peptide" is a peptide encoded by the above-mentioned foreign gene, and refers to a peptide to be produced in a protein mass production system using silk glands of silkworms. When referred to as a "peptide" in this specification, it is a molecule in which two or more amino acids are linked by amide bonds. Peptides thus include polypeptides such as oligopeptides and proteins. In addition to peptides derived from a single gene or its gene fragments, peptides also include those derived from chimeric genes obtained by linking parts of multiple genes. In addition, the amino acid length of the peptide is not particularly limited. For example, the number of amino acid residues may be 10 to 10,000. The type of the target peptide of the present specification is not particularly limited, but it is preferably a protein with high added value. Examples include peptide hormones such as insulin, calcitonin, parathyroid hormone and growth hormone, epidermal growth factor (EGF), fibroblast growth factor (FGF), interleukin (IL), interferon (IFN) , cytokines such as tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta), immunoglobulins, serum albumin, enzymes, or collagen, or fragments thereof (including chimeric peptides).

The "exogenous gene expression enhancer" refers to a drug that enhances the expression of an exogenous gene introduced into the silkworm.

"Enhancing expression" refers to increasing the expression level of a gene, especially the exogenous gene in this specification. In the present specification, expression enhancement includes an increase in the absolute amount and an increase in the relative amount of the expression level of a (exogenous) gene. Here, "increase in absolute amount" refers to an increase in the expression amount of a (exogenous) gene itself. "Increase in relative amount" means that the absolute amount of (exogenous) gene remains unchanged but the amount of recovered target peptide relatively increases due to the decrease in the expression level of other genes or the like. For example, by inhibiting gene expression of inclusion proteins such as FibH chain or sericin in silk gland, compared with when they are not inhibited, the recovery amount of the target peptide encoded by the exogenous gene is relatively increased.

"Recombinant silkworm" refers to a genetically recombinant body of silkworm having recombinant DNA produced using gene recombination technology or its progeny. The genetically recombinant silkworm of the present invention refers, in particular, to a host silkworm in which an asRNA expression vector serving as an active ingredient of the exogenous gene expression enhancer of the present embodiment is introduced into a host silkworm, or its progeny.

The "silk gland" is a tubular organ obtained by changing the salivary glands of silkworms, which are insects that can spit out silk, and has functions of producing, accumulating, and secreting liquid silk. Silk glands mainly exist as a pair of left and right along the digestive tract of silkworm larvae, and each silk gland consists of three regions: front, middle, and rear silk glands, and the middle silk gland synthesizes and secretes Sericin, and synthesize and secrete FibH chain, FibL chain and p25 in the posterior silk gland.

"Sericin (Ser)" is a water-soluble gelatin-like protein that is a coating component of silk as described above ( FIG. 1 ). It is synthesized in the middle silk gland and secreted into the lumen of the middle silk gland. Sericin is known to have a plurality of molecular species synthesized by respective genes such as Ser1, Ser2, and Ser3. However, when only "sericin" is described in this specification, it is a general term including all molecular species.

"Silk fibroin H chain (FibH)" is one of the three main proteins constituting silk fibroin which is a fiber component of silk as described above ( FIG. 1 ). It is synthesized in the posterior silk gland, forms an SFEU complex with silk fibroin L chain and p25, and is secreted into the lumen of the posterior silk gland.

1-3. Composition

The exogenous gene expression enhancer of the present embodiment contains an antisense RNA expression vector as an essential component and, if necessary, a carrier as an optional component. Hereinafter, each component is demonstrated.

1－3－1. Antisense RNA expression vector

The "antisense RNA expression vector" (in this specification, it is often referred to as "asRNA expression vector") is an active ingredient of the exogenous gene expression enhancer of the present embodiment. The mother nucleus of the asRNA expression vector can utilize various gene expression vectors. For example, expression vectors capable of autonomous replication such as plasmids or bacmids (Bacmid), viral vectors, or expression vectors capable of homologous recombination or non-homologous recombination in the chromosome, or those obtained by inserting them into the chromosome of the host part of a chromosome. In addition, the asRNA expression vector may also be a shuttle vector capable of replicating in other bacteria such as Escherichia coli.

A. Constituent elements of asRNA expression vector

The asRNA expression vector is configured to express asRNA for a target gene in the silk gland of the silkworm. The constituent elements of the expression vector contain DNA encoding at least one asRNA and a promoter required for the expression of the asRNA as essential constituent elements. In addition, other constituent elements contributing to the expression of the above-mentioned asRNA may be contained as needed. Here, other constituent elements include, for example, terminators, marker genes, enhancers, insulators, terminal inverted repeats of transposons, and the like. Furthermore, when the asRNA expression vector is composed of two units of a first subunit and a second subunit described later, DNA encoding a transcriptional regulator and a target promoter of the transcriptional regulator are included as essential constituent elements. In addition, although it is not a constituent element directly involved in the expression of asRNA, the vector may contain an exogenous gene. Hereinafter, the constitution of each element will be specifically described.

(1) DNA encoding antisense RNA (asRNA encoding DNA)

DNA encoding antisense RNA (in this specification, often referred to simply as "asRNA-encoding DNA"), encoding sericin 1-3 that will be specifically expressed in the middle silk gland of the silkworm or specifically expressed in the posterior silk gland asRNA that targets the silk fibroin H chain gene and inhibits its expression.

The asRNA (Ser1asRNA) targeting the sericin 1 (Ser1) gene targets the repeat sequence region present at positions 628 to 1054 in the full-length amino acid sequence of the silkworm Ser1 protein represented by SEQ ID NO: 1. The nucleotide sequence of Ser1asRNA contains a complementary sequence to the nucleotide sequence encoding at least one repeat unit included in the repeat region. The repeating unit of the repeating sequence region of Ser1 is composed of the amino acid sequence SX ₁ SNTDX ₂ SX ₁ X ₃ X ₄ X ₅ GSX ₆ TSGGX ₁ STYGYSSX ₇ X ₈ RX ₉ GSVSX ₂ TG represented by SEQ ID NO: 2 and a derivative sequence whose phase is changed . Here, X ₁ represents T or S, X ₂ represents S or A, X ₃ represents K or D, X ₄ represents S, N or L, X ₅ represents A or T, X ₆ represents R or S, and X ₇ represents R, S or D, X ₈ represents H, S or N, and X ₉ represents G or D in addition.

The "phase-changed derivative sequence" in the present specification refers to a sequence in which the starting amino acid position of the repeat unit is shifted in the repeat sequence region. For example, derivative sequences that alter the phase of the repeating unit represented by SEQ ID NO: 2 include sequences starting from SNT shifted from the starting amino acid position by 2 amino acids toward the C-terminal side and ending at GSX ₁ . Usually the repeating unit is continuously repeated in the repeating sequence region, so the entity of the repeating sequence does not change even when the starting amino acid of the repeating unit is shifted in its entirety. Therefore, a derivative sequence in which the phase of the repeating unit is changed can be considered to be substantially the same repeating unit as the repeating unit specified by the sequence number. Specific examples of the nucleotide sequence of Ser1asRNA include the nucleotide sequence represented by SEQ ID NO: 3. Therefore, the DNA sequence encoding the sequence represented by sequence number 3 can be one of Ser1asRNA-encoding DNAs.

The asRNA (Ser2asRNA) targeting the sericin 2 (Ser2) gene targets the repetitive sequence regions present at positions 41-117 and 685-1359 in the full-length amino acid sequence of the silkworm Ser2 protein represented by SEQ ID NO: 4 . The nucleotide sequence of Ser2asRNA contains a complementary sequence to the nucleotide sequence encoding at least one repeat unit contained in any one of the repeat regions of the two regions. The repeating units of the repeating sequence region of Ser2 are respectively present in the above two regions, the first repeating unit present at positions 41 to 117, the amino acid sequence represented by SEQ ID NO: 5 KX ₁ EX ₂ X ₃ KX ₄ X ₅ X ₆ GX ₄ and a derivative sequence that changes its phase. Here, X1 represents _F , L or V, X2 represents N or _A , _X3 represents L, I or _A , _X4 represents E or D, _X5 represents N or K, and X6 represents V or A. In addition, the amino acid sequence X ₁ SX ₂ SX ₃ X ₄ DX ₅ X ₆ KX ₇ X ₈ X ₉ X ₁₀ X ₁₁ represented by SEQ ID NO: 6 for the second repeating unit present at positions 685 to 1359 and its derivative sequence with its phase changed constitute. Here, _X1 represents G, R or S, _X2 represents S or P, _X3 represents D, H or Y, _X4 represents K or R, _X5 represents S or T, _X6 represents E or D, X ₇ represents A, V or T, X ₈ represents K or F, X ₉ represents P, H or D, X ₁₀ represents N or K, and X ₁₁ represents D, G or N. Specific examples of the nucleotide sequence of Ser2asRNA include the nucleotide sequence represented by SEQ ID NO: 7. Therefore, the DNA sequence encoding the sequence represented by sequence number 7 becomes one of Ser2asRNA encoding DNAs.

The asRNA targeting the sericin 3 (Ser3) gene (Ser3asRNA) targets the repetitive sequence regions present at positions 141-978 and 1031-1178 in the amino acid sequence of the silkworm Ser3 protein represented by SEQ ID NO: 8. The nucleotide sequence of Ser3asRNA contains a complementary sequence to the nucleotide sequence encoding at least one repeating unit included in any one of the repeating sequence regions of the two regions. The repeating units of the repeating sequence region of Ser3 are present in the above two regions respectively, and the first repeating unit present at positions 141 to 978 is represented by the amino acid sequence of SEQ ID NO: 9 SDDSSGATKGNSSKSSSSSQGX ₁ SASSSSSSX ₂ EX ₃ SSQSX ₄ SNSSNSKSSSQSSSX ₅ X ₆ NSSGSKGSGSEESSNGGSGSGRX ₇ GSX ₈ GX ₉ X ₁₀ DED and a derivative sequence composition that changes its phase. Here, X ₁ represents Q or K, X ₂ represents D or N, X ₃ represents K or N, X ₄ represents S or N, X ₅ represents S or G, X ₆ represents Q, N or K, and X ₇ represents T or N, X ₈ represents A or V, X ₉ represents G or E, and X ₁₀ represents T or S. In addition, the second repeating unit present at positions 1031 to 1178 consists of the amino acid sequence X ₁ SX ₂ SX ₃ QAX ₄ represented by SEQ ID NO: 10 and a derivative sequence whose phase is changed. Here, _X1 represents S or N, _X2 represents R or S, _X3 represents Q or K, and _X4 represents Q or H. Specific examples of the nucleotide sequence of Ser3asRNA include the nucleotide sequence represented by SEQ ID NO: 11. Therefore, the DNA sequence encoding the sequence represented by sequence number 11 can be one of Ser1asRNA-encoding DNAs.

The asRNA (Fib-HasRNA) targeting the silk fibroin H (FibH) chain gene targets the repetitive sequence region present at positions 152 to 5203 in the amino acid sequence of the silkworm FibH protein represented by SEQ ID NO: 12, and the Fib-HasRNA The base sequence contains a complementary sequence to the base sequence encoding at least one repeat unit included in the repeat region. The repeat unit in the repeat region of FibH consists of the amino acid sequence GX ₁ GX ₁ GX ₂ represented by SEQ ID NO: 13 and a derivative sequence whose phase is changed. Here, X1 represents _A , V or Y, and _X2 represents S or Y. Specific examples of the nucleotide sequence of FibHasRNA include the nucleotide sequence represented by SEQ ID NO: 14. Therefore, the DNA sequence encoding the sequence represented by sequence number 14 can be one of the DNAs encoding FibHasRNA.

(2) Promoter

The promoter acts on the asRNA-encoding DNA of the asRNA expression vector, and is an essential element for expressing the above-mentioned asRNA.

The type of promoter contained in the asRNA expression vector varies with the target gene of the asRNA. For example, when the genes of Ser1-Ser3 expressed by the middle silk gland are used as target genes, a promoter that can operate in the middle silk gland is used. Among such promoters, for example, in addition to the central silk gland-specific promoter, overexpression-type promoters, constitutively active promoters or stage-specific active promoters, or expression-inducible promoters that can be expressed generally can also be mentioned. promoter etc. A middle silk gland-specific promoter is preferable. Among them, the promoter of a gene encoding a protein that is specific to the middle silk gland and expressed in large quantities is particularly preferable. Specifically, for example, gene promoters of Ser1 to Ser3 (in this specification, referred to as "Ser1 promoter", "Ser2 promoter", and "Ser3 promoter", respectively) are mentioned. On the other hand, when the FibH chain gene expressed by posterior silk gland is used as a target gene, the promoter which can operate in posterior silk gland is used. Among such promoters, for example, in addition to the posterior silk gland-specific promoter, overexpression-type promoters, constitutively active promoters, or stage-specific active promoters that can be expressed generally, or expression-inducing type promoters, etc. A posterior silk gland-specific promoter is preferable. Among these, the promoter of the gene which encodes the protein which expresses in large quantities specifically in posterior silk gland is especially preferable. Specifically, for example, the gene promoters of FibH, FibL, or p25 (in this specification, respectively referred to as "FibH promoter", "FibL promoter" or "p25 promoter") which are proteins constituting silk fibroin .

The source organism of the promoter is not particularly limited as long as it is operable in silkworm cells into which the asRNA expression vector has been introduced. For example, a silk gland-specific promoter derived from silkworms is preferable. Usually, the nucleotide sequence of the middle silk gland-specific promoter or the rear silk gland-specific promoter is highly conserved in the evolution among silkworms, so even if the promoter used for the expression of asRNA is derived from the silkworm Different silkworms can also function in the cells of the silkworm (Sezutsu H., et al., 2009, Journal of Insect Biotechnology and Sericology, 78: 1-10). A preferred biological species of the promoter is a species belonging to the same Lepidoptera (Lepidoptera) taxonomically as the silkworm serving as a host of the asRNA expression vector, more preferably a species belonging to the same Bombycidae (Bombycidae), and even more preferably For example, species belonging to the same genus as Bombyx mandarina, most preferably Bombyx mori.

As specific examples, the following promoters can be used. Among the middle silk gland-specific promoters, the Bombyx mori-derived Ser1 promoter containing the nucleotide sequence represented by SEQ ID NO: 15, the Bombyx mori-derived Ser2 promoter containing the nucleotide sequence represented by SEQ ID NO: 16, and the sequence containing The nucleotide sequence represented by number 17 is derived from the Ser3 promoter of silkworm and the like. In addition, the Bombyx mori-derived FibH promoter containing the nucleotide sequence represented by SEQ ID NO: 18 and the tussah mori-derived FibH promoter containing the nucleotide sequence represented by SEQ ID NO: 19 can be used among the posterior silk gland-specific promoters. , the FibL promoter derived from silkworm containing the base sequence represented by SEQ ID NO: 20, the FibL promoter derived from tussah mori containing the base sequence represented by SEQ ID NO: 21, and the FibL promoter derived from silkworm containing the base sequence represented by SEQ ID NO: 22 The p25 promoter etc.

(3) terminator

The terminator consists of a base sequence capable of terminating the transcription of asRNA in the posterior silk gland cells of the host into which the asRNA expression vector is introduced.

(4) marker gene

A marker gene is a gene encoding a marker protein, also known as a selection marker. A marker protein refers to a polypeptide capable of determining the presence or absence of marker gene expression based on its activity. Therefore, by containing the marker gene in the asRNA expression vector, genetically recombinant silkworms having the asRNA expression vector can be easily determined based on the activity of the marker protein. Here "activity-based" means "activity-based detection results". The detection of the activity may be the direct detection of the activity of the labeled protein itself, or the indirect detection via metabolites such as pigments produced by the activity of the labeled protein. The detection may be any one of chemical detection (including detection of enzymatic reaction), physical detection (including detection of behavioral analysis), or sensory detection of the tester (including detection using vision, touch, smell, hearing, and taste).

The type of marker protein is not particularly limited as long as its activity can be detected by methods known in the art. Preferably, it is a marker protein that is less invasive to the genetically modified silkworm when detected. For example, fluorescent proteins, pigment-synthesizing proteins, luminescent proteins, proteins secreted externally, proteins controlling external morphology, and the like can be mentioned. Fluorescent proteins, pigment synthesis proteins, luminescent proteins, and externally secreted proteins are particularly preferred because they can be visually detected under specific conditions, and therefore have very low invasiveness to genetically modified silkworms, and are easy to judge and select.

The aforementioned fluorescent protein refers to a protein that emits fluorescence of a specific wavelength when the genetically engineered silkworm is irradiated with excitation light of a specific wavelength. It may be any of natural type and non-natural type. In addition, the excitation wavelength and fluorescence wavelength are not particularly limited. Specifically, examples include CFP, AmCyan, RFP, DsRed (including derivatives such as DsRedmonomer and DsRed2), YFP, GFP (including derivatives such as EGFP and EYFP), and the like.

The above-mentioned pigment synthesis protein is a protein involved in the biosynthesis of pigment, and is usually an enzyme. The term "pigment" here refers to a low-molecular compound or peptide capable of imparting a pigment to a transformant, and its type is not limited. It is preferably a pigment that expresses as an individual external color. For example, melanin-based pigments (including dopamine melanin), eye pigment-based pigments, or pteridine-based pigments are mentioned.

The aforementioned luminescent protein refers to a substrate protein capable of luminescence without excitation light or an enzyme that catalyzes the luminescence of the substrate protein. For example, aequorin and luciferase as an enzyme are mentioned.

In the present specification, an externally secreted protein refers to a protein secreted outside the cell or outside the body, and belongs to exocrine enzymes and the like. Exocrine enzymes include enzymes that contribute to the decomposition or inactivation of agents such as blasticidin and impart drug resistance to the host, as well as digestive enzymes.

The marker gene is placed in an expressible state downstream of the above-mentioned promoter in the asRNA expression vector. The promoter used may be the same or different from the asRNA-encoding DNA.

(5) Insulators

An insulator is a sequence that stably controls the transcription of a gene sandwiched by the sequence without being affected by the chromatin of the surrounding chromosome. For example, chicken cHS4 sequence, Drosophila gypsy sequence, etc. are mentioned.

(6) Terminal inverted repeat sequence of transposon

The inverted terminal repeat sequence (Inverted terminal repeat sequence) of the transposon (HandlerAM.etal., 1998, Proc.Natl.Acad.Sci.U.S.A.95:7520-5) can be contained when the asRNA expression vector is an asRNA expression vector capable of recombination . Terminal inverted repeats are arranged upstream and downstream of the asRNA expression vector. As the transposon, piggyBac, mariner, minos and the like can be used (Shimizu, K. et al., 2000, Insect Mol. Biol., 9, 277-281; Wang W. et al., 2000, Insect Mol Biol 9(2): 145-55).

(7) DNA encoding transcriptional regulators

"DNA encoding a transcriptional regulator" is an essential element of the first subunit described later, and refers to a gene of a transcriptional regulator. The term "transcriptional regulator" in this specification refers to a protein factor that binds to a target promoter described later and is capable of activating the target promoter. For example, GAL4 protein, which is a galactose metabolism activation protein of yeast, and tTA, a tetracycline-controlled transcriptional activator, and its mutants, etc., can be mentioned.

(8) Target promoters of transcriptional regulators

"Target promoter of a transcriptional regulator" is an essential element of the second subunit described later, and refers to a promoter capable of activating the expression of a certain gene under the control of the binding of the transcriptional regulator encoded by the first subunit . The above-mentioned transcriptional regulators and their target promoters correspond to the above-mentioned transcriptional regulators. Generally, if a transcriptional regulator is identified, its target promoter can also be identified. For example, when the transcription regulator is GAL4 protein, UAS (Upstream Activating Sequence) is used.

(9) Exogenous genes

Exogenous gene, as described in the above definition, is an exogenous gene that can be expressed in the silk gland and encodes a target peptide. The original function of the asRNA expression vector contained in the exogenous gene expression enhancer of this embodiment is to express asRNA that suppresses the expression of target genes such as Ser1 to Ser3 and FibH chain. Therefore, the exogenous gene encoding the target peptide has no direct relationship with the composition of the asRNA expression vector. However, the purpose of asRNA is to indirectly enhance the expression of exogenous genes by inhibiting the expression of the above-mentioned target genes, and efficiently produce target peptides. That is, an exogenous gene is an object necessary for the exogenous gene expression enhancer of the present embodiment to exert its effect. Therefore, when the host insect which administers the exogenous gene expression enhancer of this embodiment does not have the exogenous gene expressible in silk gland, the object of this invention cannot fully be achieved. The exogenous gene encoding the target peptide is usually contained in another gene expression vector (exogenous gene expression vector) described later, and introduced into the same host insect independently of the asRNA expression vector. However, it may also be included in an asRNA expression vector in a state capable of expressing the target exogenous gene. When the asRNA expression vector contains an exogenous gene, in order to express the exogenous gene in the silk gland, the above-mentioned middle silk gland-specific promoter and/or posterior silk gland-specific promoter can be arranged upstream of it.

B. The unit composition of asRNA expression vector

The asRNA expression vector includes a case composed of one gene expression unit and a case composed of two gene expression units. Each case is explained below.

(1) In the case of one gene expression unit

When the asRNA expression vector consists of one gene expression unit, as long as an exogenous gene expression enhancer containing the asRNA expression vector is administered to the silkworm, the asRNA of interest can be expressed in the silkworm cell. Such an asRNA expression vector contains all the constituent elements necessary for the expression of asRNA in one unit. That is, it includes at least one expression system consisting of a promoter as an essential element and an asRNA-encoding DNA functionally bound downstream of the promoter. This set of expression systems may be a system containing one asRNA-encoding DNA under the control of one promoter, or may be a system containing two or more asRNA-encoding DNAs.

(2) In the case of two gene expression units (Fig. 2)

When the asRNA expression vector is composed of two gene expression units, the first subunit and the second subunit, the constituent elements necessary for the expression of asRNA exist separately in the two units. Therefore, as long as these two exist in the silkworm cell, they can function as one asRNA expression vector and express the desired asRNA. Each subunit has the following constitution.

The first subunit comprises a promoter and a DNA encoding the above-mentioned transcription regulator functionally bound downstream of the promoter. 2A-C illustrate the first subunit. As the promoter used in the first subunit, a promoter capable of functioning in the silk gland of silkworm silkworm was used, similarly to the above-mentioned promoter controlling the expression of the asRNA-encoding DNA. For example, the gene promoters of proteins that are specific and synthesized in large quantities in the posterior silk glands such as FibH, FibL or p25 shown in the posterior silk gland promoter shown in Figure 2A, such as the middle silk gland shown in Figure 2B The silk gland promoter, such as sericin protein, is a gene promoter of a protein that is specifically synthesized in a large amount in the middle silk gland. As shown in Figure 2C, there are multiple sets of promoters and DNA encoding transcription regulators in the first subunit, and each promoter can be set as a rear silk gland promoter and a middle silk gland promoter. Such a different combination. In addition, in FIGS. 2A to 2C , the GAL4 gene is exemplified as DNA encoding a transcription regulator.

The second subunit comprises the target promoter of the transcription regulator encoded by the first subunit and an asRNA-encoding DNA functionally bound downstream of the target promoter. Figures 2D and E show an example of the second subunit. In Fig. 2D, UAS is exemplified as a target promoter of GAL4. In addition, the second subunit, in addition to the system containing one asRNA-encoding DNA under the control of one target promoter as shown in Figure 2D, may also contain two asRNAs under the control of one target promoter as shown in Figure 2E. The above asRNA-encoded DNA system. It should be noted that when the asRNA expression vector contains an exogenous gene encoding the target peptide, it is preferably included in the second subunit.

The asRNA expression vector of this constitution functions as a set of first and second subunits as described above. Through the activation of the silk gland expression promoter, the transcription regulator expressed by the first subunit activates the target promoter of the second subunit, thereby expressing the target asRNA. In addition, the second subunit may be composed of two or more subunits containing the same or different asRNA-encoding DNAs. In this case, the transcription regulator expressed by one first subunit can express asRNA encoded by each second subunit by activating the target promoters of multiple second subunits.

The asRNA expression vector of this configuration can amplify the expression of the asRNA-encoding DNA of the second subunit via the transcription regulator encoded by the first subunit. Therefore, when the expression ability of the silk gland expression promoter of the 1st subunit is not high, it is especially preferable.

In the asRNA expression vector of this configuration, only the asRNA-encoding DNA region of the second subunit may be exchanged with another asRNA-encoding DNA region in an expression frame, and the first subunit may be used in common. That is, the first subunit can also utilize an existing gene expression vector or a gene recombinant silkworm system introduced therein. Therefore, the asRNA expression vector consisting of two gene expression units is also convenient in terms of production cost and labor of the expression unit and gene recombinant silkworm.

It should be noted that when the asRNA expression vector is composed of two subunits, the first subunit and the second subunit, the exogenous gene expression enhancer of the present embodiment is, in principle, composed of the first subunit containing the first subunit as an active ingredient. The exogenous gene expression enhancer and the second exogenous gene expression enhancer containing the second subunit as an active ingredient are constituted as a set of two doses. As described above, since the first subunit can be used together, the first exogenous gene expression enhancer and the second exogenous gene expression enhancer do not necessarily need to be in a one-to-one correspondence, and the second exogenous gene expression enhancer can also be obtained. One exogenous gene expression enhancer is used as a co-agent, and only the second exogenous gene expression enhancer is exchanged for a configuration of each combination of 2 doses per set. In addition, if the host insect administered with the exogenous gene expression enhancer of this embodiment is a genetically recombined insect that already has the first subunit in the chromosome, then the exogenous gene expression enhancer of this embodiment can be obtained only by combining The second subunit corresponding to the first subunit is constituted as a second exogenous gene expression enhancer as an active ingredient.

1－3－2. Carrier

The exogenous gene expression enhancer of the present invention can contain an acceptable carrier as needed. "Acceptable carriers" include solvents, adjuvants and the like generally used in the field of entomology.

Among the solvents, for example, water, an aqueous solution, or an organic solvent acceptable to silkworms can be mentioned. Examples of aqueous solutions include buffer solutions (phosphate buffer, sodium acetate buffer, etc.), physiological saline, and isotonic solutions. Typically, water or an aqueous solution is used.

Among the adjuvants, for example, auxiliary carriers are mentioned. A helper vector, for example, contains DNA encoding a transposon transferase. When the asRNA expression vector as an active ingredient of the exogenous gene expression enhancer has the terminal inverted repeat sequence of the transposon, the auxiliary vector can realize the insertion of the asRNA expression vector into the chromosome of the host insect by producing transposon transferase. As the helper vector, if the host insect is silkworm, for example, pHA3PIG can be mentioned. Examples of other auxiliary agents include glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acids Esters, etc.

2. Gene recombinant silkworm

2－1. Summary

The second embodiment of the present invention is genetically recombinant silkworm. The genetically recombinant silkworm of the present embodiment is characterized by comprising the asRNA expression vector described in the first embodiment above. When the gene-recombined silkworm of this embodiment further has an exogenous gene expression vector, the asRNA expressed from the asRNA expression vector suppresses the expression of endogenous genes such as sericin and FibH chain which are its targets. Thus, the target peptide encoded by the exogenous gene can be efficiently produced in the silk gland of the silkworm.

2－2. Composition

The genetically recombinant silkworm of the present invention has the asRNA expression vector described in the first embodiment above. The structure of the asRNA expression vector is the same as that of the asRNA expression vector described in the first embodiment, so its description will be omitted. Here, the structure unique to the genetically modified silkworm of the present embodiment will be described.

In the genetically modified silkworm, the asRNA expression vector according to the first embodiment may exist temporarily in the silkworm cell, or may persist stably in a state of being introduced into a chromosome or the like. Generally, stable and persistent are preferred.

The genetically recombinant silkworm can have two or more different asRNA expression vectors described in the first embodiment. For example, genetically recombinant silkworms may have both an expression vector consisting of one gene expression unit and an expression vector consisting of two gene expression units. In this case, each asRNA expression vector may contain the same or different asRNA-encoding DNA.

As described above, the gene expression unit consists of two subunits, the first subunit and the second subunit, and when each exists on the chromosome of the silkworm, each subunit may exist on the same chromosome or on a different chromosome. When each subunit exists on a different chromosome, by recombining the silkworm system with only the gene of the first subunit (preferably homozygous) and the gene recombination of the silkworm with only the second subunit (preferably homozygous) By mating, the recombinant silkworm of the present invention having the first subunit and the second subunit can be easily obtained in F1. In this case, the above-mentioned recombinant silkworm system having only the first subunit can be commonly used for mating with various recombinant silkworm systems having only the second subunit as described in the first embodiment.

On the other hand, when the first subunit and the second subunit exist on the same chromosome, the distance between the subunits is preferably short and they are associated with each other so that they do not separate from each other due to recombination during passage.

The genetically recombinant silkworm of this embodiment does not necessarily need to contain an exogenous gene encoding a target peptide. As described above, this is because the gene-recombined silkworm of the present embodiment already has the function of suppressing the expression of the target gene by having an asRNA expression vector. However, the object of the present invention, which is to increase the production efficiency of the target peptide encoded by the exogenous gene in the silk gland of silkworm silkworm, cannot be achieved if it has only the asRNA expression vector. In order to improve the production efficiency of the target peptide in the silk gland of the silkworm, the gene-recombined silkworm of the present embodiment requires the presence of an exogenous gene that is the object to exhibit the function. Therefore, when using the genetically modified silkworm of the present embodiment as a protein mass production system, in addition to the asRNA expression vector, it is necessary to further have an expression system (exogenous gene expression vector) encoding an exogenous gene encoding the target peptide. The exogenous gene expression vector can be prepared in such a manner that the genetically recombinant silkworm of the present embodiment is kept in advance as needed. It should be noted that when the asRNA expression vector contains an exogenous gene encoding the target peptide, the genetically recombinant silkworm of this embodiment can also achieve the purpose of the present invention by having the asRNA expression vector.

2－3. Production method

The method for producing the genetically modified silkworm of the present embodiment is only a method capable of producing a recombinant silkworm expressing the desired asRNA from the expression vector described in the first embodiment above, and any method can be adopted without any particular limitation. As a method of producing such a recombinant silkworm, for example, a method of administering the exogenous gene expression enhancer according to the first embodiment described above to a silkworm as a host, and adding the gene expression enhancer described in the first embodiment on a different chromosome The method for mating silkworms with gene recombination of the first subunit and the second subunit of the asRNA expression vector.

The method of administering the exogenous gene expression enhancer of the first embodiment to silkworms can be performed by a method known in the art capable of introducing an asRNA expression vector, which is an active ingredient of the exogenous gene expression enhancer, into silkworms. For example, when administering the exogenous gene expression enhancer of the first embodiment to silkworm eggs, the method of Tamura et al. (Tamura T. et al., 2000, Nature Biotechnology, 18, 81-84) for introducing a gene expression vector into silkworm can be used. . Specifically, an administration solution is prepared by dissolving or diluting an exogenous gene expression enhancer with a solvent such as water or a buffer so that the contained asRNA expression vector becomes an appropriate concentration. Here, when the asRNA expression vector has a transposon terminal inverted repeat sequence at both ends of the asRNA-encoding DNA, a helper vector having a DNA encoding a transposon transferase can be added to the administration solution and injected into the early developmental stage of the silkworm egg. The silkworm used at this time may be a wild-type silkworm, or a genetically recombined silkworm with an exogenous gene expression vector that produces the target peptide in the silk gland of the silkworm. In addition, when the exogenous gene expression enhancer contains a helper carrier, the above-mentioned administration solution can be directly injected into the silkworm eggs at the early stage of development. Then, by selecting transformants based on the selection marker, the recombinant silkworm with the gene of interest can be obtained. In the genetically recombined silkworm obtained by the method, the asRNA expression vector in the exogenous gene expression enhancer is assembled into the chromosome through the terminal inverted repeat sequence of the transposon. Sibling mating or homologous mating of the gene recombinant silkworm can be carried out as required to obtain a homozygote of the expression vector inserted into the chromosome.

On the other hand, the method of producing the genetically recombinant silkworm of the present invention by mating the genetically recombinant silkworms having the first subunit and the second subunit of the expression vector described in the first embodiment on different chromosomes can also be carried out by this method. methods known in the art. For example, first, the first exogenous gene expression enhancer and the second exogenous gene expression enhancer containing the respective subunits are administered to individual silkworms, respectively. The method of administration can be the method of introducing the gene expression vector into the silkworm as above, for example, the method of Tamura et al. In this case, for example, genetically recombinant silkworms having a first subunit on their chromosomes have already been established and can be used, and only the expression of the second exogenous gene containing a second subunit corresponding to the first subunit can be enhanced. The agent is introduced into the silkworm. For example, the genetically recombined silkworm silkworm having the first subunit in which the gene encoding the GAL4 protein is arranged downstream of the middle silk gland promoter such as the sericin promoter in the chromosome has been established, and when it can be used, only the first subunit can be used. 2. Administering an exogenous gene expression enhancer to silkworms to produce genetically recombinant silkworms having a second subunit on their chromosomes, the second exogenous gene expression enhancer containing the downstream of the UAS promoter that is the target sequence of the GAL4 protein The second subunit of target asRNA encoding DNA is used as an active ingredient. The obtained gene-recombined silkworms having each subunit are mated with siblings or inbred, and each subunit is preferably made homozygous in advance. Next, by mating the gene-recombined silkworms with each subunit, and selecting F1 individuals with two subunits, the target gene-recombined silkworm can be obtained. Selection was performed based on the respective selection markers for the 1st and 2nd subunits.

After producing the genetically recombinant silkworm of this embodiment, an exogenous gene expression vector can be further introduced as necessary. At this time, the method of introducing the exogenous gene expression vector into the genetically recombinant silkworm of the present embodiment can be carried out by the same method as above.

3. Peptide Manufacturing Method

3－1. Summary

The third embodiment of the present invention is a method for producing a peptide. According to the production method of the present embodiment, the target peptide encoded by the exogenous gene can be efficiently produced in the silk gland of the gene-recombined silkworm while suppressing the expression of sericin, FibH chain, and other contaminating proteins.

3－2. Materials

The peptide production method of the present embodiment uses the genetically modified silkworm as a protein mass production system. The genetically recombinant silkworm used in this embodiment is a genetically recombinant silkworm having the asRNA expression vector described in the first embodiment and the exogenous gene expression vector. That is, in other words, it is the genetically recombinant silkworm of the above-mentioned second embodiment having an exogenous gene expression vector.

The exogenous gene expression vector possessed by the genetically recombinant silkworm used in this embodiment contains an exogenous gene encoding a target peptide to be produced in the peptide production method of this embodiment.

3-3. Manufacturing method

The production method of the present invention includes a breeding step and a recovery step. Hereinafter, each step will be described.

(1) Feeding process

The "breeding process" is a process of rearing genetically modified silkworms. Regarding the breeding method of the genetically recombinant silkworm, it can be raised according to the breeding techniques of the silkworm known in the art. For example, you can refer to "General Discussion on Silkworm Eggs; Work by Takami Husband, Journal of the National Silkworm Egg Association". The feed can use natural leaves of herbivorous tree species such as Morus leaves, or artificial feed such as SilkMate L4M or the 1-3 instars of silkworm eggs (Japanese agricultural industry). From the viewpoint of suppressing disease occurrence, providing bait with stable quality and quantity, and enabling aseptic breeding if necessary, artificial feed is preferable. Hereinafter, an example of a simple method of raising genetically modified silkworms will be described.

The operation of moving and raising young silkworms to silkworm beds is performed using eggs laid by females of silkworms of an appropriate number (for example, 4 to 10) of the recombinant silkworm. The hatched larvae are transferred from silkworm egg paper to a container covered with anti-drying paper (paraffin processed paper) as a silkworm seat, and artificial feeds such as SilkMate are placed on the anti-drying paper to provide bait. In principle, the exchange of bait is carried out once at the 1st to 2nd age, and 1 to 3 times at the 3rd age. When there is a lot of old bait leftover, it is removed in order to prevent corruption. When the healthy silkworm larvae of the 4th to 5th instar are reared, they are transferred to a large container, and the number of each container is appropriately adjusted. Depending on the humidity and the state of the container, the container may be covered with a lid made of anti-drying paper, acrylic resin, or wire mesh. The feeding temperature is kept at 25-28°C throughout the whole age.

(2) Recycling process

The "recovery process" is a process of recovering the target peptide accumulated in the silk gland of the larvae of the genetically modified silkworm.

The gene-recombined silkworm used in this embodiment mainly expresses an exogenous gene from the late last instar of the larvae, and the encoded target peptide is accumulated in the silk gland cells. The method for recovering the target peptide from the genetically recombinant silkworm is not limited. For example, there is a method in which the silk gland is extracted from the insect body from the late last instar to the prepupal stage, and the target peptide is directly recovered. The specific method can be realized by methods known in the art. For example, after anesthetizing silkworms that are about to spin silk on the 6th day of the last age (5th instar) on ice, the dorsal side is incised and extracted with a pin or the like in a manner that does not damage the silk glands (refer to Yasushi Mori, compiled using New Biological Experiments of Silkworm, Sanseido, 1970, pp.249-255). The extracted silk gland can be slowly shaken at a temperature of, for example, 0 to 10° C., preferably 0 to 5° C., in the above-mentioned extraction buffer, so that the target peptide can be dissolved in the buffer. If the target peptide is not a thermosensitive peptide, it can be carried out at a temperature of 10-40°C. Then, impurities such as tissue pieces are removed by centrifugation or filtration, and the supernatant containing the target peptide is collected.

Example

<Example 1: Inhibition of Ser1 gene expression by Ser1asRNA>

(Purpose)

It was demonstrated that the gene expression of sericin 1 (Ser1), which is a protein specific to the middle silk gland, can be suppressed by asRNA directed against the Ser1 gene.

(method)

1. Production of a specific expression vector for the silk gland in the middle of the silkworm

As the expression vector that silk gland in the silkworm middle part can specifically express, make the Ser1asRNA expression vector expressing the asRNA (referred to as " Ser1asRNA ") to Ser1 gene and express the shRNA (represented as " Ser1shRNA " to Ser1 gene. It should be explained, The shRNA in this specification also includes the Ser1 shRNA expression vector having a longdsRNA with a hairpin structure.

(1) Preparation of UAS backbone plasmid

PCR amplification was performed using pAmCyan1-N1 (Takara) as a template and AmCyankozakU (SEQ ID NO: 23) and SV40PCRL (SEQ ID NO: 24) as a primer pair, and inserted into pZErO-2 vector (lifetechnologies). This was referred to as AmCyan/pZero2.

In addition, PCR amplification was performed using pEYFP-N1 (clonetech) as a template, and EYFPkozakU (SEQ ID NO: 25) and SV40PCRL as a primer pair, and inserted into pZErO-2 vector (lifetechnologies). This was designated as EYFP/pZero2.

AmCyan/pZero2 and EYFP/pZero2 were cleaved using NcoI-NotI, and the resulting NcoI-NotI fragment was inserted into the NcoI of pBac[SerUAS/3xP3EGFP] (TatematsuK.etal., 2010, TransgenicResearch, 19(3):473-87) - Notl site whereby the marker gene is replaced by AmCyan or EYFP from EGFP. These plasmids were designated as pBac[SerUAS/3xP3AmCyan] or pBac[SerUAS/3xP3EYFP].

(2) Cloning of Ser1 fragment

Total RNA (IsogenNipponGene) was extracted from the middle silk gland of the 5th instar and 6th day of the Bombyx mori white/C system. Total RNA was reverse-transcribed using oligodTprimer (Promega) and reverse transcriptase (ReverTraAceTOYOBO) to obtain cDNA. Using the obtained cDNA as a template, PCR was performed using two primer pairs, SpeSer1B130U23 (SEQ ID NO: 26) and BlnSer1B1636L23 (SEQ ID NO: 27), and BlnSer1B130U23 (SEQ ID NO: 28) and SpeSer1B1636L23 (SEQ ID NO: 29), to obtain the 5' end of Ser1. fragment. These nucleic acid fragments are referred to as 5'Ser1SB and 5'Ser1BS.

In addition, using the obtained cDNA as a template, PCR was performed using two primer pairs of SpeSer1B1531U24 (SEQ ID NO: 30) and BlnSer1B3108L23 (SEQ ID NO: 31), and BlnSer1B1531U24 (SEQ ID NO: 32) and SpeSer1B3108L23 (SEQ ID NO: 33) to obtain Ser1 3' fragments. These nucleic acid fragments are referred to as 3'Ser1SB and 3'Ser1BS.

(3) Preparation of shRNA linker

A linker corresponding to the loop portion of the shRNA is made. The genomic DNA of the silkworm white/C system was used as a template, and the primer pair used BlnA3intU (SEQ ID NO: 34) and SpeA3IntL (SEQ ID NO: 35) to perform PCR amplification on the intron of actin 3. The obtained nucleic acid fragment was used as linker A3intBS.

(4) Production of Ser1asRNA expression vector

5'Ser1SB was inserted into the BlnI site of pBac[SerUAS/3xP3AmCyan] prepared in (1) above so that BlnI became the 5' end. This plasmid was designated as pBac[SerUAS-5'asSer1/3xP3AmCyan] (Fig. 3A). pBac[SerUAS-5'asSer1/3xP3AmCyan] is a 5'Ser1asRNA expression vector that expresses 5'Ser1asRNA targeting the 5' region of the Ser1 gene that does not contain a repeat sequence region.

In addition, 3'Ser1SB was inserted into the BlnI site of pBac[SerUAS/3xP3EYFP] so that BlnI becomes the 5' end. This plasmid was designated as pBac[SerUAS-3'asSer1/3xP3EYFP] (Fig. 3B)). pBac[SerUAS-3'asSer1/3xP3EYFP] is a 3'Ser1asRNA expression vector that expresses a 3'Ser1asRNA targeting the repeat sequence region present at the 3' end of the Ser1 gene. The 3'Ser1asRNA expression vector corresponds to the second subunit of the asRNA expression vector containing the exogenous gene expression enhancer of the present invention as an active ingredient.

(5) Production of Ser1shRNA expression vector

The linker A3intBS was inserted into the BlnI site of the 5'Ser1asRNA expression vector so that the BlnI site became the 5' end, and the 5'Ser1BS was further inserted so that the BlnI site became the 5' end. This plasmid was designated as pBac[SerUAS-5'dsSer1/3xP3AmCyan] (Fig. 4A). pBac[SerUAS-5'dsSer1/3xP3AmCyan] is a 5'Ser1shRNA expression vector expressing 5'Ser1shRNA targeting the 5' region of the Ser1 gene.

In addition, the linker A3intBS was inserted into the BlnI site of the 3'Ser1asRNA expression vector so that the BlnI site became the 5' end, and the 3'Ser1BS was further inserted so that the BlnI site became the 5' end. This plasmid was designated as pBac[SerUAS-3'dsSer1/3xP3EYFP] (Fig. 4B). pBac[SerUAS-3'dsSer1/3xP3EYFP] is a 3'Ser1shRNA expression vector that expresses a 3'Ser1shRNA targeting the repeat sequence region present at the 3' end of the Ser1 gene.

2. Production of genetically recombined silkworm

(1) Introduction of expression vector

The Ser1asRNA expression vectors pBac[SerUAS-5'asSer1/3xP3AmCyan], pBac[SerUAS-3'asSer1/3xP3EYFP] and Ser1shRNA as the Ser1asRNA expression vectors produced in the above "1. Production of silk gland-specific expression vectors in the middle part of the silkworm", respectively. The expression vector pBac[SerUAS-5'dsSer1/3xP3AmCyan] and pBac[SerUAS-3'dsSer1/3xP3EYFP] were individually combined with the helper plasmid pHA3PIG expressing transposon transferase (TamuraT.etal., 2000, Nature Biotechnology, 18,81 -84) mixed at a ratio of 1:1 and injected into silkworm eggs of the w1-pnd system 2 to 8 hours after oviposition. The injected eggs were incubated at 25°C in a humidified state until hatching. The hatched larvae were reared for all ages in a breeding room at 25-27° C. using artificial feed (SilkMate original species 1-3 instar S, Nippon Agricultural Industry). The artificial feed was exchanged every 2-3 days (Uchino K. et al., 2006, JInsect Biotechnol Sericol, 75:89-97). After eclosion, sibling mating takes place. The genetically recombinant silkworm line was obtained from the F1 eggs obtained by selection based on the presence or absence of eye fluorescence caused by the 3xP3EYFP marker or the 3xP3AmCyan marker as the respective selection markers.

(2) Mating with the GAL4 system of the middle silk gland

Each gene recombination silkworm system obtained by the above "(1) introduction of expression vector" was systematically combined with the pBacSer-proGAL4/3xP3DsRed2 (TatematsuK.etal., 2010, TransgenicResearch, 19(3) shown in Figure 5 :473-87) genetically recombined silkworm system mating. Here, pBacSer-proGAL4/3xP3DsRed2 is a central silk gland GAL4 expression vector that specifically expresses the GAL4 gene in the central silk gland, and corresponds to the first asRNA expression vector containing the exogenous gene expression enhancer of the present invention as an active ingredient. subunit. In the F1 individual, according to the presence or absence of eye fluorescence caused by the 3xP3EYFP marker or the 3xP3AmCyan marker and the 3xP3DsRed2 marker, the Ser1asRNA expression vector or the Ser1shRNA expression vector (the second subunit) and the pBacSer-proGAL4/3xP3DsRed2 (the first subunit) were selected. subunit) system to obtain the gene recombinant silkworm silkworm specifically expressing Ser1asRNA or Ser1shRNA in the middle silk gland. Since each 3 system uses the genetically recombined silkworm with the second subunit, the genetically recombined silkworm that specifically expresses Ser1asRNA or Ser1shRNA in the middle silk gland is also obtained from each 3 system.

3. Quantification of Ser1 mRNA

The effect of suppressing the expression of the Ser1 gene in the middle silk gland of each genetically recombined silkworm was verified using the recombined silkworm in which Ser1asRNA or Ser1shRNA was specifically expressed in the middle silk gland produced in the above-mentioned "2. Production of genetically modified silkworm".

The middle silk gland was extracted from the larvae of the fifth instar and the sixth day of each gene-recombined silkworm, and total RNA was extracted using ISOGEN (Nippon Gene). Total RNA was reverse-transcribed using oligodT primers (Promega) and reverse transcriptase (ReverTraAceTOYOBO) to prepare cDNA. Using the obtained cDNA as a template, quantitative PCR of Ser1 mRNA was performed with LightCyclerFastStartDNAMasterSYBRGreen (Roche) using the primer pair of Ser1LC3364 (SEQ ID NO: 36) and Ser1LC3528L (SEQ ID NO: 37). A primer pair of rp49LCF2 (SEQ ID NO: 38) and rp49LCR1 (SEQ ID NO: 39) was used as the internal standard, and quantitative PCR was also performed.

4. Verification of Ser1 protein quantity

It was confirmed that the synthesis of Ser1 protein was also suppressed at the protein level using the genetically engineered silkworm which expresses Ser1asRNA or Ser1shRNA specifically in the middle part silk gland produced by said "2. Preparation of genetically modified silkworm". Recombinant silkworms of each gene were bred to obtain cocoons, and 1 mL of 50 mM Tris-HCl (PH8.0)/8M urea/10 mMDTT was added to 10 mg of cocoons and incubated at 80° C. for 5 minutes to extract cocoon proteins. 12.5 μL of NuPAGELDSampleBuffer (Invitrogen) and 5 μL of NuPAGESampleReducingAgent (Invitrogen) were added to 32.5 μL of cocoon protein, and heated at 70° C. for 10 minutes for SDS. The SDS-formed sample was electrophoresed on a 4% SDS-PAGE gel, and stained with EZstainaqua (ATTO).

(result)

The results of the above "3. Quantification of Ser1 mRNA" are shown in Fig. 6 . This figure shows relative values when the amount of Ser1 mRNA transcription in the negative control genetically modified silkworm (the system represented by "-" in the second subunit of FIG. 6 ) is 1. Any group of recombinant silkworms having a 5'Ser1shRNA expression vector, genetically recombinant silkworms having a 3'Ser1shRNA expression vector, genetically recombinant silkworms having a 5'Ser1asRNA expression vector, and genetically recombinant silkworms having a 3'Ser1asRNA expression vector were confirmed to inhibit Expression of the Ser1 gene. The average relative value of the Ser1 mRNA amount of the 3 systems (corresponding to the second subunits 1, 2, and 3) of each group is shown in the upper part.

Among asRNAs that are composed of single-stranded RNA and do not form an intramolecular double-stranded RNA region, the group expressing 5'Ser1asRNA showed lower Ser1 expression than the two groups expressing shRNA that formed an intramolecular double-stranded RNA region. The expression suppression effect of the gene was very low. On the other hand, the group expressing 3'Ser1asRNA targeting the repeat sequence region of Ser1 had an effect of suppressing Ser1 gene expression more than shRNA despite being asRNA. This result shows that the asRNA targeting the repeat region of the Ser1 protein effectively suppresses the gene expression of the Ser1 protein in the middle silk gland compared with the case of targeting other regions.

The results of the above "4. Verification of Ser1 protein amount" are shown in FIG. 7 . The position of each cocoon protein is shown on the right side of SDS-PAGE. The Ser1 protein is represented by alternative splicing as two bands with different molecular weights. The genetically recombinant silkworm system with only the first subunit (UAS-; GAL4+) and the genetically recombinant silkworm system with no first subunit but only the second subunit (UAS3; GAL4-) were used as controls.

The same results were obtained at the protein level as in the experiments at the mRNA level. That is, it was confirmed that the synthesis of Ser1 protein was inhibited in all genetically recombinant silkworms other than the control. On the other hand, the synthesis amount of the Ser3 protein and the FibH chain protein which are the same cocoon proteins was about the same as that of the control. This indicates that asRNA or shRNA expressed by Ser1 asRNA expression vector or shRNA expression vector specifically inhibits the expression of Ser1 gene.

<Example 2: Suppression of FibH gene expression by asRNA against FibH chain gene>

(Purpose)

Example 1 demonstrates the expression suppression effect of the asRNA for the gene of the middle part silk gland specific protein Ser1. Therefore, this Example proves that the expression of the FibH gene can also be suppressed by using asRNA directed against the repeat region of the FibH gene for the silk fibroin H (FibH) chain which is the posterior silk gland-specific protein.

(method)

1. Production of specific expression vectors for the posterior silk gland of the silkworm

A Fib-HasRNA expression vector expressing asRNA for the FibH chain gene was prepared as an expression vector capable of specific expression in the posterior silk gland of the silkworm.

(1) Preparation of UAS backbone plasmid

BsmBIadapU (SEQ ID NO: 40) and BsmBIadapL (SEQ ID NO: 41) were annealed to make a BsmBI linker. A BsmBI linker was inserted into the BlnI site of pBac[SerUAS/3xP3AmCyan] produced in Example 1. The obtained plasmid was designated as pBac[SerUAS-BsmBI/3xP3AmCyan].

(2) Cloning of the repeat region of the FibH chain

The primer pair of FibHU (SEQ ID NO: 42) and FibHL (SEQ ID NO: 43) was used to perform PCR on the genomic DNA of the silkworm large-scale production system to amplify the repeat sequence region of the FibH chain gene. Two fragments selected from the obtained various fragments were designated as FibH1 and FibH2, respectively. FibH1 (1196bp) and FibH2 (1433bp) were cleaved with BsmBI, and an antisense strand was inserted into the BsmBI site of pBac[SerUAS-BsmBI/3xP3AmCyan] in the direction of expression. The resulting plasmids were designated as pBac[SerUAS-asFib1/3xP3AmCyan] ( FIG. 8A ) and pBac[SerUAS-asFib2/3xP3AmCyan] ( FIG. 8B ), respectively. These plasmids are all Fib-HasRNA expression vectors containing asRNA (referred to as "Fib-HasRNA") encoding DNA for the repeat sequence region of the FibH chain gene, which is equivalent to containing the exogenous gene expression enhancer of the present invention as an active ingredient. The first subunit of the asRNA expression vector.

2. Production of recombinant silkworm

(1) Introduction of expression vector

Mix pBac[SerUAS-asFib1/3xP3AmCyan] and pBac[SerUAS-asFib2/3xP3AmCyan] with the helper plasmid pHA3PIG at a ratio of 1:1, and inject them into silkworm eggs of the w1-pnd system 2 to 8 hours after oviposition. The injected eggs were incubated at 25°C in a humidified state until hatching. The hatched larvae were reared for all ages in a breeding room at 25-27° C. using artificial feed (SilkMate original species 1-3 instar S, Nippon Agricultural Industry Co., Ltd.). The artificial feed was exchanged every 2-3 days. After eclosion, sibling mating takes place. According to the presence or absence of eye fluorescence caused by 3xP3AmCyan, the obtained F1 eggs were selected to obtain a gene recombinant silkworm system. For the genetically recombined silkworms of 2 groups of FibH1 and FibH2, 2 systems and 3 systems were respectively selected for mating with the subsequent posterior silk gland GAL4 system.

(2) Mating with the posterior silk gland GAL4 system

The genetically recombinant silkworm system obtained above was mated with the genetically recombinant silkworm system having pBac[BmFibHL-GAL4/3xP3-DsRed] (Sezutsu H. et al, 2009, Journal of Insect Biotechnology and sericology, 78, 1-10). pBac[BmFibHL-GAL4/3xP3-DsRed] (Fig. 9) is the posterior silk gland GAL4 expression vector that is connected with GAL4 gene in the downstream of the FibH promoter that has activity in the posterior silk gland, and is equivalent to containing the present invention. The first subunit of the asRNA expression vector in which the exogenous gene expression enhancer is used as an effective component. In F1 individuals, the Fib-HasRNA expression vector (second subunit) and pBac[BmFibHL-GAL4/3xP3-DsRed] (first subunit) were selected based on the presence or absence of eye fluorescence caused by the 3xP3AmCyan marker and the 3xP3DsRed2 marker. ) both systems, the rear silk gland specifically expresses the gene recombinant silkworm for the FibH chain asRNA.

3. Quantification of FibH chain mRNA

The effect of suppressing the expression of the FibH chain gene of the posterior silk gland was verified using the genetically recombinant silkworm which expresses Fib-HasRNA specifically in the posterior silk gland produced in said "2. Production of genetically recombinant silkworm".

The posterior silk glands were extracted from the 5th and 6th day larvae of each gene-recombined silkworm, and total RNA was extracted using ISOGEN (Nippon Gene). Total RNA was reverse-transcribed using oligodT primers (Promega) and reverse transcriptase (ReverTraAceTOYOBO) to prepare cDNA. Using the obtained cDNA as a template, quantitative PCR of FibH chain mRNA was performed with LightCyclerFastStartDNAMasterSYBRGreen (Roche) using a primer pair of FibHLCU (SEQ ID NO: 44) and FibHLCL (SEQ ID NO: 45). The primer pair of rp49LCF2 and rp49LCR1 was used as the internal standard, and quantitative PCR was also performed.

4. Verification of FibH chain protein quality

Using the genetically engineered silkworm that expresses Fib-HasRNA specifically in the posterior silk gland produced in the above-mentioned "2. Production of genetically modified silkworm", it was confirmed that the synthesis of FibH chain protein was suppressed also at the protein level. The silk glands were extracted from the 5th and 6th day larvae of each gene-recombined silkworm, and separated into posterior silk glands and middle silk glands. Add each posterior silk gland or middle silk gland to 5 mL of 20mM Tris-HCl (pH8.0)/8M urea/2% SDS/25mMDTT, shake overnight at room temperature, and extract silk gland protein. Although the FibH chain gene is specifically expressed in the posterior silk gland, the reason for the verification of the protein in the middle silk gland is because the FibH chain protein is synthesized in the posterior silk gland and transferred to the middle silk gland, so not only Present in the posterior silk gland and also in the middle silk gland. 27.5 μL of _H2O , 12.5 μL of NuPAGELDSampleBuffer (Invitrogen) and 5 μL of NuPAGESampleReducingAgent (Invitrogen) were added to 5 μL of silk gland protein, and heated at 70° C. for 10 minutes for SDS. The SDS-formed sample was electrophoresed on a 4% SDS-PAGE gel, and then stained with EZstainaqua (ATTO).

(result)

The results of the above "3. Quantification of FibH chain mRNA" are shown in FIG. 10 . This figure shows relative values when the amount of FibH chain mRNA transcription in the control genetically modified silkworm (the system in which the second subunit in FIG. 10 is indicated by "-") is 1. The expression inhibition of FibH chain gene was confirmed in the recombinant silkworm FibH1 and FibH2 with Fib-HasRNA expression vector. This result shows that the asRNA directed to the repeat region of the silk gland protein of Bombyx mori is effective not only for the middle silk gland-specific protein but also for the posterior silk gland-specific protein.

The results of the above "4. Verification of FibH chain protein quantity" are shown in FIG. 11 . The positions of FibH chain protein and Ser1 protein are shown on the right side of SDS-PAGE. The white/C system is a non-recombinant silkworm that becomes the basis of the genetically recombinant silkworm of this example. The Nd system is a mutation system of silkworms with low expression of FibH chain genes, and the Nd-sD system is a mutation system of silkworms with low expression of FibL chain genes. In the Nd system and the Nd-sD system, the expression of both FibH chain and FibL chain genes decreased due to the degeneration of the posterior silk gland. Gene recombinant silkworm system (UAS-; GAL4+) is a silkworm that only has the first subunit (GAL4) and does not have the second subunit (UAS-FibHasmRNA). These are controls for this example.

The same results were obtained at the protein level as in the experiments at the mRNA level. That is, in the genetically recombinant silkworm having two subunits of the Fib-HasRNA expression vector, the amount of FibH chain protein in the middle silk gland of any system was significantly reduced compared with the control (UAS-; GAL4+). This shows that the synthesis of FibH chain protein by the expression of Fib-HasRNA in the posterior silk gland is inhibited, and no new FibH chain protein is synthesized, and finally the posterior silk gland lumen is extruded to the middle silk gland lumen FibH chain protein quality decreased.

On the other hand, Ser1 protein, which is the same silk gland protein, was synthesized in the middle silk gland at the same level as the control (UAS-; GAL4+), and Fib expressed by the Fib-HasRNA expression vector was also shown at the protein level. -HasRNA specifically inhibits the expression of Fib-H chain genes.

<Example 3: Inhibition of Ser1 and FibH chain gene expression using Ser1asRNA and Fib-HasRNA>

(Purpose)

It was proved that the combination of asRNA targeting the repeat sequence region of each gene of Ser1 and FibH can simultaneously inhibit the expression of Ser1 gene and FibH chain gene when expressed simultaneously.

(method)

1. Production of expression vectors for the middle silk gland and posterior silk gland of the silkworm

(1) Production of Fib-HasRNA and Ser1asRNA simultaneous expression UAS vector (UAS-3'Ser1-FibH1asRNA expression vector)

The 3'Ser1SB prepared in Example 1 was inserted into the BlnI site of pBac[SerUAS-asFibH1/3xP3AmCyan] to create pBac[SerUAS-3'asSer1_asFibH1/3xP3AmCyan] (Fig. 12A). The plasmid is an expression vector (UAS-3'Ser1-FibH1asRNA expression vector) in which 3'Ser1asRNA-encoded DNA and Fib-HasRNA-encoded DNA are configured in series downstream of the UAS promoter, which is equivalent to containing the exogenous gene expression enhancer of the present invention The second subunit of the asRNA expression vector as an active ingredient.

(2) Production of middle & posterior silk gland GAL4 expression vector

AscI.adU (SEQ ID NO: 46) and AscI.adL (SEQ ID NO: 47) were annealed to prepare an AscI linker. The AscI linker was inserted into the AscI site of pBac[BmFibHL-GAL4/3xP3-DsRed] shown in Fig. 9 to prepare pBacFibH-proGAL4_AscI-BlnI/3xP3DsRed2. Further insert the AscI-SpeI fragment of pBacSer-proGAL4/3xP3DsRed2 into the AscI-BlnI site of this plasmid, and make the middle & back silk gland GAL4 expression vector pBacFibH-proGAL4_Ser that expresses the GAL4 gene in both the middle and back silk glands - proGAL4/3xP3DsRed2 (Fig. 12B). This expression vector corresponds to the first subunit of the asRNA expression vector containing the exogenous gene expression enhancer of the present invention as an active ingredient.

2. Production of recombinant silkworm

(1) Introduction of expression vector

The pBacFibH-proGAL4_Ser-proGAL4/3xP3DsRed2 which is the GAL4 expression vector of the middle & posterior silk gland and the pBac[SerUAS-3'asSer1_asFibH1/3xP3AmCyan] which is the UAS-3'Ser1-FibH1asRNA expression vector are respectively described in Example 1 The helper plasmid pHA3PIG was mixed at a ratio of 1:1 and injected into silkworm eggs of the w1-pnd system 2 to 8 hours after oviposition. The injected eggs were incubated at 25°C in a humidified state until hatching. The hatched larvae were reared for all ages in a breeding room at 25-27° C. using artificial feed (SilkMate original species 1-3 instar S, Nippon Agricultural Industry). The artificial feed was exchanged every 2-3 days. After eclosion, sibling mating takes place. From the obtained F1, the middle & posterior silk gland GAL4 system injected with the middle & posterior silk gland GAL4 expression vector was selected according to the presence or absence of eye fluorescence caused by the 3xP3DsRed2 marker, and also based on the 3xP3AmCyan marker The UAS-3'Ser1-FibH1asRNA system with the UAS-3'Ser1-FibH1asRNA expression vector was selected and injected with the presence or absence of fluorescence of the eyes to obtain a genetically recombined silkworm system.

(2) Mating of middle & posterior silk gland GAL4 system and UAS-3'Ser1-FibH1asRNA system

The middle & posterior silk gland GAL4 system was mated with the UAS-3'Ser1-FibH1asRNA system. In addition, as a control, the gene recombination silkworm system with the middle silk gland GAL4 expression vector pBacSer-proGAL4/3xP3DsRed2 used in Example 1 or the rear silk gland GAL4 expression vector pBac[BmFibHL-GAL4 used in Example 2 /3xP3-DsRed] gene recombinant silkworm system was mated with the Ser1-FibH1asRNA system. In the F1 individual, the system having both the central & posterior silk gland GAL4 system and the UAS-3'Ser1-FibH1asRNA system was selected according to the presence or absence of eye fluorescence caused by the 3xP3DsRed2 marker and the 3xP3AmCyan marker, and the central silk was obtained The genetically recombined silkworm silkworm expressing Fib-HasRNA and Ser1asRNA simultaneously in gland and/or posterior silk gland.

3. Verification of Ser1 protein quantity and FibH chain protein quantity

The Ser1 protein quantity and the FibH chain protein quantity were verified using the genetically recombinant silkworm produced in the above "2. Production of genetically recombinant silkworm". The silk glands were extracted from the 5th and 6th day larvae of each gene-recombined silkworm, and separated into posterior silk glands and middle silk glands. Add each posterior silk gland or middle silk gland to 5 mL of 20mM Tris-HCl (pH8.0)/8M urea/2% SDS/25mMDTT, shake overnight at room temperature, and extract silk gland protein. Add 27.5 μL of H ₂ O, 12.5 μL of NuPAGELDSampleBuffer (Invitrogen) and 5 μL of NuPAGESampleReducing Agent (Invitrogen) to 5 μL of silk gland protein, and heat at 70° C. for 10 minutes for SDS. The SDS-formed sample was electrophoresed on a 4% SDS-PAGE gel, and then stained with CBC.

(result)

Figure 13 shows the results. When the GAL4 vector for posterior silk gland expression was used for the 1st subunit, the FibH chain protein amount of the middle silk gland decreased compared with the control (UAS-; GAL4+). This is consistent with the result of Example 2, and when the first subunit uses the GAL4 vector for expression of the middle silk gland, the Ser1 protein of the middle silk gland almost disappears. This indicates that Ser1asRNA expression in the middle silk gland suppresses the expression of the Ser1 gene by the GAL4 vector for expression in the middle silk gland. On the other hand, when the first subunit uses the GAL4 vector for expression of the middle & posterior silk gland, Fib-HasRNA and Ser1asRNA are expressed in both the middle and the posterior silk gland. Thus, the expression of both the Ser1 gene and the FibH chain gene is simultaneously and efficiently suppressed.

<Example 4: Using Ser1asRNA to enhance the expression of exogenous genes>

(Purpose)

According to Examples 1 and 3, it was demonstrated that Ser1 gene expression was suppressed by Ser1asRNA. Therefore, it was demonstrated that the expression of exogenous genes can be enhanced by suppressing the expression of the Ser1 gene.

(method)

1. Production of Exogenous Gene Expression Vectors

The UAS backbone plasmid AmCyan/pZero2 produced in Example 1 was cut with NcoI-NotI, and the AmCyan gene fragment was cut out. The resulting fragment was inserted into the NcoI-NotI site of pBac[SerUAS-ser_int-EGFP/3xP3EGFP] ( FIG. 14A ) (Tatematsu K. et al., 2010, Transgenic Research, 19(3):473-87). Thus, the marker gene of pBac[SerUAS-ser_int-EGFP/3xP3EGFP] was replaced by AmCyan from EGFP. The obtained UAS-EGFP expression vector was designated as pBacSerUAS-ser_intEGFP/3xP3AmCyan ( FIG. 14B ).

2. Production of recombinant silkworm

(1) Introduction of exogenous gene expression vector

The UAS-EGFP expression vector pBacSerUAS-ser_intEGFP/3xP3AmCyan prepared in the above "1. Production of exogenous gene expression vector" was mixed with the helper plasmid pHA3PIG described in Example 1 at a ratio of 1:1, and injected until 2 Bombyx mori eggs of the w1-pnd system for ~8 hours. The injected eggs were incubated at 25°C in a humidified state until hatching. The hatched larvae were reared for all ages in a breeding room at 25-27° C. using artificial feed (SilkMate original species 1-3 instar S, Nippon Agricultural Industry). The artificial feed was exchanged every 2-3 days. After eclosion, sibling mating takes place. From the F1 selected based on the presence or absence of eye fluorescence caused by the 3xP3AmCyan marker, a genetically recombinant silkworm system was obtained. The genetically recombined silkworm system was designated as the UAS-EGFP system.

(2) Mating of GAL4 system, 3'Ser1asRNA system and UAS-EGFP system in the middle silk gland

The middle silk gland GAL4 system (equivalent to the first subunit system) having pBacSer-proGAL4/3xP3DsRed2 as the GAL4 vector for middle silk gland expression and the 3'Ser1asRNA expression vector pBac[SerUAS-3'asSer1/3xP3EYFP] The UAS-3'SerasRNA system (equivalent to the second subunit system) was mated with the UAS-EGFP system having pBacSerUAS-ser_intEGFP/3xP3AmCyan obtained in "(1) Introduction of exogenous gene expression vector". For comparison, the above-mentioned central silk gland GAL4 system was mated with the UAS-3'SershRNA system having the 3'Ser1shRNA expression vector pBac[SerUAS-3'dsSer1/3xP3EYFP] and the above-mentioned UAS-EGFP system. In addition, as a negative control, only the above-mentioned middle silk gland GAL4 system and the above-mentioned UAS-EGFP system were crossed. Based on the markers of the respective expression vectors, a system having three types of gene expression vectors for a single individual or a system for negative controls having two types of gene expression vectors was selected according to the presence or absence of eye fluorescence. Thereby, the system which expresses EGFP gene specifically and suppresses the expression of Ser1 gene in the middle part silk gland, and the system which does not generate|occur|produce the expression suppression of Ser1 gene were obtained.

3. Quantification of EGFP Protein in Silk Gland

The amount of EGFP protein was verified using each gene-recombined silkworm produced in the above-mentioned "2. Production of genetically-recombined silkworm". The middle silk glands were extracted from the 5th day 6th instar larvae, each one was added to 10 mL of PBS (pH7.2)/1% Tween20/0.05% sodium azide, and the water-soluble protein was extracted by shaking at room temperature for 24 hours. The obtained water-soluble protein extract was centrifuged at 2000×g for 10 minutes, and the supernatant was collected. The EGFP protein concentration in the water-soluble protein contained in the supernatant was measured using Reacti-BindAnti-GFPCoated Plates (PIERCE). Specifically, add 100 μL of supernatant to Reacti-BindAnti-GFPCoatedPlates and let stand at room temperature for 1 hour. After washing 3 times with PBS/0.05% Tween20, horseradishperoxidase-conjugated anti-GFPantibody (Rockland Immunochemicals) was added and left to stand at room temperature for 1 hour. After washing three times with PBS/0.05% Tween20, a color reaction was performed using TMBPeroxidase EIA Substrate Kit (Bio-Rad), and 1N sulfuric acid was added to stop the reaction. Color development was quantified using a platereader (SpectraMax250; Molecular Devices). A standard curve was prepared using serial dilutions (1-400 pg/μL) of recombinant GFP protein (TAKARABIO; Z2373N).

(result)

Fig. 15 shows the amount of EGFP protein per individual of each system. Compared with the control ("-" in Fig. 15 ), the system expressing 3'Ser1asRNA had about 2-fold increase in the amount of EGFP protein. This shows that the expression of Ser1 gene is suppressed by expressing 3' Ser1asRNA directed at the repeat sequence region of Ser1 gene in the middle silk gland, and finally the expression of exogenous gene (here, EGFP gene) is enhanced. On the other hand, inhibition of Ser1 gene expression by 3'Ser1shRNA also increased the amount of EGFP protein, but the amount of increase was lower than that of 3'Ser1asRNA.

<Example 5: Enhancing the expression of exogenous genes by the expression of Ser1asRNA and Fib-HasRNA>

(Purpose)

It was verified that the expression of exogenous genes can be further enhanced by expressing Ser1asRNA and Fib-HasRNA in combination and simultaneously inhibiting the expression of Ser1 gene and FibH gene.

(method)

1. Production of recombinant silkworm

The middle & back silk gland GAL4 system, UAS-3'Ser1-FibH1asRNA system and the UAS prepared in Example 4 with the middle & back silk gland GAL4 expression vector pBacFibH-proGAL4_Ser-proGAL4/3xP3DsRed2 prepared in Example 3 -EGFP system for mating. In addition, as a control, only the middle & posterior silk gland GAL4 system and the UAS-EGFP system were crossed. Select according to the presence or absence of eye fluorescence caused by the markers of each expression vector, and obtain a genetic recombination in which the silk gland specifically expresses EGFP and the middle silk gland and/or posterior silk gland simultaneously express Fib-HasRNA and Ser1asRNA There are 3 systems for each silkworm.

2. Quantification of EGFP Protein in Silk Gland

The amount of EGFP protein was verified using each gene-recombined silkworm produced in the above-mentioned "1. Production of genetically-recombined silkworm". The silk glands were extracted from the 5th day 6th instar larvae, each one was added to 20 mL of PBS (pH7.2)/1% Tween20/0.05% sodium azide, and the water-soluble protein was extracted by shaking at room temperature for 24 hours. The obtained water-soluble protein extract was centrifuged at 2000×g for 10 minutes, and the supernatant was collected. The EGFP protein concentration in the water-soluble protein contained in the supernatant was measured using Reacti-BindAnti-GFPCoated Plates (PIERCE). Specifically, add 100 μL of supernatant to Reacti-BindAnti-GFPCoatedPlates and let stand at room temperature for 1 hour. After washing 3 times with PBS/0.05% Tween20, horseradishperoxidase-conjugated anti-GFPantibody (Rockland Immunochemicals) was added and left to stand at room temperature for 1 hour. After washing three times with PBS/0.05% Tween20, a color reaction was performed using TMBPeroxidase EIA Substrate Kit (Bio-Rad), and 1N sulfuric acid was added to stop the reaction. Color development was quantified using a platereader (SpectraMax250; Molecular Devices). A standard curve was prepared using serial dilutions (1-400 pg/μL) of recombinant GFP protein (TAKARABIO; Z2373N).

(result)

Figure 16 shows the amount of EGFP protein per individual for each system. In the system expressing 3'Ser1asRNA and Fib-HasRNA, compared with the negative control which did not suppress the expression of these genes ("-" of FIG. 17), the high EGFP protein amount was found in silk gland. This result indicated that the expression of an exogenous gene (here, EGFP gene) was enhanced by the inhibition of the synthesis of Ser1 and FibH chains, which are the main proteins of the middle silk gland and the posterior silk gland, by the respective asRNAs.

<Example 6: Reduction of inclusion proteins by expression of Ser1asRNA and Fib-HasRNA>

(Purpose)

It was confirmed that gel-like inclusion proteins (mainly composed of sericin and silk fibroin) that hinder the extraction of the target peptide can be reduced by expressing Ser1asRNA and Fib-HasRNA.

(method)

The middle part and the back silk gland were extracted from each 3 larvae of the 5th instar 6th day of the same system as the gene recombinant silkworm produced in Example 5. One aliquot of the middle and posterior silk glands was collected and added to 4 mL of LPBS with inverting stirring overnight at 4°C. Centrifuge at 5000 rpm at 4°C, put the obtained supernatant into a 15 mL tube, and let stand at 4°C for 48 hours. The tube was turned upside down, the amount of liquid dropped was measured, and the percentage of liquid dropped relative to the original amount of liquid was calculated.

(result)

Figure 17 shows the results. In the negative control in which the expression of Ser1 and FibH chain genes was not suppressed, the supernatant gelled and became cloudy in white. Finally, even if the tube was turned upside down, the extract did not drop at all. On the other hand, in the samples derived from the system expressing Ser1asRNA and Fib-HasRNA, the supernatant did not gel and remained in a liquid state. When the tube was turned upside down, an average of 98% of the extract fell out. These results showed that by suppressing the expression of the Ser1 gene and the FibH chain gene by Ser1asRNA and Fib-HasRNA, respectively, the inclusion protein which becomes the cause of the gelation of the silk gland extract can be removed effectively.

In addition, all publications, patents, and patent applications cited in this specification are incorporated in this specification as it is by reference.

Claims (12)

1. An exogenous gene expression enhancer of the silk gland of silkworm, characterized in that, it contains the silk gland specific expression in the middle part of silkworm silkworm selected from sericin 1 antisense RNA, sericin 2 antisense RNA and The antisense RNA expression vector of at least one antisense RNA in the sericin 3 antisense RNA and/or the antisense RNA expression vector capable of specifically expressing the silk fibroin H chain antisense RNA in the posterior silk gland of the silkworm as an effective ingredients, of which, The antisense RNA of sericin 1 contains a sequence complementary to the base sequence of at least one repeating unit in the repeating sequence region from position 628 to position 1054 in the amino acid sequence encoding silkworm sericin 1 shown in sequence number 1, The sericin 2 antisense RNA contains the base of at least one repeating unit in the repeating sequence region of positions 41-117 or 685-1359 in the amino acid sequence of silkworm sericin 2 shown in the coding sequence number 4 Sequences that are complementary to each other, The sericin 3 antisense RNA contains the base of at least one repeating unit in the repeat sequence region of positions 141-978 and positions 1031-1178 in the amino acid sequence of silkworm sericin 3 shown in the coding sequence number 8 Sequences that are complementary to each other, The silk fibroin H-chain antisense RNA contains a sequence complementary to the base sequence of at least one repeating unit in the repeat sequence region from position 152 to position 5203 in the amino acid sequence of the silk fibroin H chain shown in sequence number 12 . 2. The exogenous gene expression enhancer according to claim 1, wherein the repeating unit of sericin 1 is composed of the amino acid sequence shown in sequence number 2, The repeating unit of sericin 2 consists of the amino acid sequence shown in sequence number 5 or 6, The repeating unit of sericin 3 consists of the amino acid sequence shown in sequence number 9 or 10, in addition The repeating unit of silk fibroin H chain consists of the amino acid sequence shown in SEQ ID NO:13. 3. The exogenous gene expression enhancer according to claim 1, wherein, Sericin 1 antisense RNA consists of the base sequence shown in SEQ ID NO: 3, Sericin 2 antisense RNA consists of the base sequence shown in SEQ ID NO: 7, Sericin 3 antisense RNA consists of the base sequence shown in SEQ ID NO: 11, and in addition The silk fibroin H-chain antisense RNA consists of the base sequence shown in SEQ ID NO:14. 4. The exogenous gene expression enhancer according to any one of claims 1 to 3, wherein the antisense RNA expression vector consists of two subunits. 5. A genetically recombined silkworm, characterized in that it has the ability to specifically express the silk gland in the middle part of the silkworm selected from sericin 1 antisense RNA, sericin 2 antisense RNA and sericin 3 antisense RNA. An antisense RNA expression vector of at least one antisense RNA and/or a silk fibroin H chain antisense RNA expression vector capable of specifically expressing antisense RNA in the posterior silk gland of the silkworm, wherein, The antisense RNA of sericin 1 contains a sequence complementary to the base sequence of at least one repeating unit in the repeating sequence region from position 628 to position 1054 in the amino acid sequence encoding silkworm sericin 1 shown in sequence number 1, The sericin 2 antisense RNA contains the base of at least one repeating unit in the repeating sequence region of positions 41-117 or 685-1359 in the amino acid sequence of silkworm sericin 2 shown in the coding sequence number 4 Sequences that are complementary to each other, The sericin 3 antisense RNA contains the base of at least one repeating unit in the repeat sequence region of positions 141-978 or 1031-1178 in the amino acid sequence of silkworm sericin 3 shown in the coding sequence number 8 Sequences that are complementary to each other, The antisense RNA specifically expressed by the rear silk gland of the silkworm contains the base of at least one repeat unit in the repeat sequence region of position 152 to position 5203 in the amino acid sequence of the silk fibroin H chain shown in the coding sequence number 12 The sequence complementary to the base sequence. 6. The gene recombinant silkworm according to claim 5, wherein, The repeating unit of sericin 1 consists of the amino acid sequence shown in SEQ ID NO: 2, The repeating unit of sericin 2 consists of the amino acid sequence shown in sequence number 5 or 6, The repeating unit of sericin 3 consists of the amino acid sequence shown in sequence number 9 or 10, or The repeating unit of silk fibroin H chain consists of the amino acid sequence shown in SEQ ID NO:13. 7. The gene recombinant silkworm according to claim 5, wherein, Sericin 1 antisense RNA consists of the base sequence shown in SEQ ID NO: 3, Sericin 2 antisense RNA consists of the base sequence shown in SEQ ID NO: 6, Sericin 3 antisense RNA consists of the base sequence shown in SEQ ID NO: 9, or The silk fibroin H-chain antisense RNA consists of the base sequence shown in SEQ ID NO:14. 8. The genetically recombinant silkworm according to any one of claims 5 to 7, wherein the antisense RNA expression vector consists of 2 subunits. 9. The genetically recombinant silkworm according to claim 8, wherein the two subunits are present on different chromosomes. 10. The gene recombinant silkworm according to any one of claims 5 to 9, having an exogenous gene expression vector capable of expressing a target exogenous gene. 11. A method for producing gene-recombined silkworm, characterized by comprising the step of administering the exogenous gene expression enhancer according to any one of claims 1 to 4 to silkworm. 12. A method for producing the target peptide encoded by an exogenous gene in the silk gland of the genetically recombined silkworm of claim 10.