CN105683373A

CN105683373A - Exogenous gene expression enhancer

Info

Publication number: CN105683373A
Application number: CN201480058676.4A
Authority: CN
Inventors: 立松谦郎; 立松谦一郎; 濑筒秀树; 内野惠郎; 小岛桂
Original assignee: National Institute of Agrobiological Sciences
Current assignee: National Institute of Agrobiological Sciences
Priority date: 2013-10-25
Filing date: 2014-10-14
Publication date: 2016-06-15
Also published as: JPWO2015060159A1; WO2015060159A1; JP6326703B2

Abstract

The purpose is to develop and provide a method for easily and efficiently suppressing gene expression of sericin and/or fibroin H-chain, which are major silk gland-specific proteins, to efficiently produce a target protein in the silk glands as a protein mass production system in genetically-modified silkworms. Provided is a silkworm line capable of expressing within the silkworm antisense RNA having as a target site a repetitive sequence region of sericin and/or fibroin H-chain.

Description

Expression of exogenous genes reinforcing agent

Technical field

The present invention relates to a kind of in the protein expression system that make use of silkworm silk glands using the antisense RNA expression vector expression of exogenous genes reinforcing agent as effective ingredient, and there is the gene recombinaton silkworm of this antisense RNA expression vector.

Background technology

The silk glands of silkworm (Bombyxmori), has the ability of a large amount of protein of short time synthesis. Further, since the silk glands of silkworm be large-scale organ so easily extracting, the protein of synthesis is stored in silk glands inner chamber, so the advantage also with easily recovery. Therefore, utilize silk glands to express the gene recombinaton silkworm of target protein, be considered to hold promise as the mass procution system of the high protein of the added value such as useful proteins matter or high-performance silk.

Silkworm silk glands, morphology is the organ of 1 pair, left and right as shown in Figure 1, is made up of anterior silk glands, middle part silk glands and 3 regions of rear portion silk glands respectively. In the silk glands cell of middle part, synthesize the covering water miscible gelatin sample protein of composition, sericin 1 as spun silk (in this specification, usually referred to as " Ser1 "), sericin 2 is (in this specification, usually referred to as " Ser2 ") and sericin 3 (in this specification, usually referred to as " Ser3 ") (Fig. 1). These protein, are secreted in the silk glands inner chamber of middle part in post synthesis. Additionally, in the silk glands cell of rear portion, synthesize 3 main protein and fibroin albumen H chain that constitute the fibroin albumen belonging to silk flock composition (in this specification, usually referred to as " FibH "), fibroin albumen L chain is (in this specification, usually referred to as " FibL ") and p25/FHX (following, to be designated as " p25 "). These 3 protein form complex (silkfibroinelementaryunit with the ratio of FibH:FibL:p25=6:6:1; In this specification, hereinafter referred to as " SFEU complex "), secretion is to the silk glands inner chamber of rear portion. Then, SFEU complex is transferred to middle part silk glands inner chamber, is covered by sericin, weaves silk (Fig. 1: non-patent literature 1) as spun silk from anterior silk glands. Therefore, using silkworm silk glands as protein expression system utilize time, utilize specific expressed gene expression system in middle part silk glands or rear portion silk glands. Actually, so far as the recombinant protein expression system using middle part silk glands, establish the great expression method (non-patent literature 3) of system utilizing GAL4/UAS system (non-patent literature 2) or Ser1 promoter and Hr3 enhancer being combined.

In the protein expression system utilizing silkworm silk glands, in order to improve the production efficiency of target protein, improve the expression of this gene itself, by reducing other major protein quality of silk glands so that the expression of target protein rises relatively and be important from silkworm efficient recovery target protein. Hinder one of reason of Protein Recovery, the existence being mingled with protein can be enumerated. Such as, the fibroin albumen of the main protein as silk glands, sericin can be enumerated. These protein are many due to synthetic quantity, and are fiber qualities, and toughness, crystallinity are high, therefore easily become the reason of blocking post or filter when target protein extracts or refines. Therefore, for the efficient recovery of protein, it is necessary to suppress these to be mingled with the gene expression of protein. On the other hand, the excessive expression inhibiting of these genes expressed in silkworm silk glands, owing to can produce impact to the growth of silkworm itself, it is therefore desirable to control the technology of this expression inhibiting.

In silkworm silk glands, it is suppressed that in the method for the expression of endogenous fibroin albumen or sericin, including the method for the method and the artificial expression suppressing these genes that use existing gene mutation system.

As the example of method using existing gene mutation system, it is known that the FibH chain expressed by rear portion silk glands in silkworm has abnormal Nd system, FibL chain has abnormal Nd-s system and Nd-sD system. Due in Nd system FibH chain express or in Nd-s system and Nd-sD system the expression of FibL chain non-normally low, therefore by using these systems to utilize middle part silk glands to make target protein expression can improve its expression (patent documentation 1). But, said gene abruptly-changing system, it is impossible to the gene expressions such as the Ser1 that suppression is expressed by middle part silk glands, it addition, the abruptly-changing system etc. of the gene expressed by the middle part silk glands of silkworm is not reported so far.

As the example of method of the expression of artificial suppressor gene, RNAi (RNAinteference) method can be enumerated. RNAi method is the decomposition of transcription product via target gene in vivo, it is suppressed that the method for the expression of (silence) this gene. Generally in RNAi method, use the siRNA (smallinterferingRNA) that is made up of double-stranded RNA or miRNA (microRNA) and be made up of the RNAi molecule such as the shRNA (shorthairpinRNA) that formed double-stranded region by autofolding (self-folding) in molecule single stranded RNA. On the other hand, it is made up of single stranded RNA and is formed without the antisense RNA in intramolecular duplex RNA region (in this specification, usually be designated as " asRNA ") with said target mrna annealing, eventually pass through and double-stranded RNA identical process play the expression inhibiting effect of the gene caused by RNAi. But, owing to its effect is non-normally low compared with other RNAi molecule, therefore it is substantially free of use (non-patent literature 4).

In Lepidoptera (Lepidoptera) insecticide headed by silkworm, the effect generally low (non-patent literature 5) of gene expression inhibition caused by RNAi compared with other biological kind. However, it is known that its effect is different because organizing difference, even Lepidoptera, also simply relatively high in skin or silk glands. Such as, the report double-stranded RNA cell inner expression silkworm by making FibH chain, FibL chain and Ser1, it is possible to suppress the expression (patent documentation 2～4) of each gene. But, these methods, owing to must express and construct double-stranded RNA in vivo, and the complex procedures that the making of expression vector or gene recombinaton silkworm make needs labour, therefore there is the problem that manufacturing cost uprises. And, in the prior art, exist and combine multiple genes and suppress it to express extremely difficult problem simultaneously. These problems, if the asRNA being made up of single stranded RNA then can overcome. But, the effect reporting in silk fabric gland of being in gene expression inhibition caused by asRNA is also non-normally low, is only about one (non-patent literature 6) of hundreds of points compared with double-stranded RNA.

Prior art literature

Patent documentation

Patent documentation 1: Japanese Unexamined Patent Publication 2004-135528

Patent documentation 2: Japanese Unexamined Patent Application Publication 2006-521802

Patent documentation 3: Japanese Unexamined Patent Publication 2008-245623

Patent documentation 4: Japanese Unexamined Patent Publication 2011-103816

Non-patent literature

Non-patent literature 1:InoueS.etal., 2000, TheJournalofBiologicalChemistry, 275 (51): 40517-40528.

Non-patent literature 2:TatematsuK.etal., 2010, TransgenicResearch, 19 (3): 473-87.

Non-patent literature 3:TomitaM.etal., 2007, TransgenicResearch, 16 (4): 449-465.

Non-patent literature 4:TavernarakisN.etal., 2000, Naturegenetics, 24180-183.

Non-patent literature 5:TereniusO.etal., 2011, JournalofInsectPhysiology, (57): 231-245

Non-patent literature 6:IsobeR.etal., 2002, Journalofinsectbiotechnologyandsericology, 71,43-47

Summary of the invention

The problem of the present invention is in that exploitation and provides effective suppression as the method for the endogenic main protein of silk glands and the gene expression that can become the sericin being mingled with protein and/or FibH chain, to improve the production efficiency of target protein in the silk glands of gene recombinaton silkworm.

In order to solve above-mentioned problem, the present inventor etc. further investigate, finally found that by make using sericin, FibH chain aminoacid sequence in the repetitive sequence region contained express in the silk glands of silkworm as the asRNA of direct target region, even if thus asRNA also is able to effectively suppress the gene expression of sericin, FibH chain. The present invention is based on this achievement in research, it is provided that invention below.

(1) the expression of exogenous genes reinforcing agent of a kind of silkworm silk glands, it contains can make silk glands in the middle part of silkworm specific expressed selected from Ser1asRNA, the asRNA expression vector of at least one asRNA and/or the asRNA expression vector of the silkworm rear portion specific expressed Fib-HasRNA of silk glands can be made as effective ingredient in Ser2asRNA and Ser3asRNA, wherein, Ser1asRNA contains the sequence of base sequence complementary of at least one repetitive in the repetitive sequence region of 628～1054 in the aminoacid sequence of the silkworm Ser1 represented with coded sequence numbering 1, Ser2asRNA contains the sequence of base sequence complementary of at least one repetitive in the repetitive sequence region of 41～117 or 685～1359 in the aminoacid sequence of the silkworm Ser2 represented with coded sequence numbering 4, Ser3asRNA contains the sequence of base sequence complementary of at least one repetitive in the repetitive sequence region of 141～978 or 1031～1178 in the aminoacid sequence of the silkworm Ser3 represented with coded sequence numbering 8, Fib-HasRNA contains the sequence of base sequence complementary of at least one repetitive in the repetitive sequence region of 152～5203 in the aminoacid sequence of the silkworm FibH chain represented with coded sequence numbering 12.

(2) the expression of exogenous genes reinforcing agent recorded according to (1), wherein, the aminoacid sequence that the repetitive of Ser1 is represented by sequence numbering 2 is constituted, the aminoacid sequence that the repetitive of Ser2 is represented by sequence numbering 5 or 6 is constituted, the aminoacid sequence that the repetitive of Ser3 is represented by sequence numbering 9 or 10 is constituted, and the aminoacid sequence that additionally repetitive of FibH chain is represented by sequence numbering 13 is constituted.

(3) the expression of exogenous genes reinforcing agent recorded according to (1), wherein, the base sequence that the asRNA of Ser1 is represented by sequence numbering 3 is constituted, the base sequence that the asRNA of Ser2 represents due to sequence numbering 7 is constituted, the base sequence that the asRNA of Ser3 is represented by sequence numbering 11 is constituted, and the base sequence that additionally FibH chain asRNA is represented by sequence numbering 14 is constituted.

(4) the expression of exogenous genes reinforcing agent recorded any one of (1)～(3), wherein, above-mentioned asRNA expression vector is made up of 2 subunits.

(5) a kind of gene recombinaton silkworm, have and silk glands in the middle part of silkworm can be made specific expressed selected from Ser1asRNA, the asRNA expression vector of at least one asRNA and/or the asRNA expression vector of the silkworm rear portion specific expressed Fib-HasRNA of silk glands can be made in Ser2asRNA and Ser3asRNA, wherein, Ser1asRNA contains the sequence of base sequence complementary of at least one repetitive in the repetitive sequence region of 628～1054 in the aminoacid sequence of the silkworm Ser1 represented with coded sequence numbering 1, Ser2asRNA contains the sequence of base sequence complementary of at least one repetitive in the repetitive sequence region of 41～117 or 685～1359 in the aminoacid sequence of the silkworm Ser2 represented with coded sequence numbering 4, Ser3asRNA contains the sequence of base sequence complementary of at least one repetitive in the repetitive sequence region of 141～978 or 1031～1178 in the aminoacid sequence of the silkworm Ser3 represented with coded sequence numbering 8, Fib-HasRNA contains the sequence of base sequence complementary of at least one repetitive in the repetitive sequence region of 152～5203 in the aminoacid sequence of the silkworm FibH chain represented with coded sequence numbering 12.

(6) the alia gene recombinant silkworm recorded according to (5), wherein, the aminoacid sequence that the repetitive of Ser1 is represented by sequence numbering 2 is constituted, the aminoacid sequence that the repetitive of Ser2 is represented by sequence numbering 5 or 6 is constituted, the aminoacid sequence that the repetitive of Ser3 is represented by sequence numbering 9 or 10 is constituted, and the aminoacid sequence that additionally repetitive of FibH chain is represented by sequence numbering 13 is constituted.

(7) the gene recombinaton silkworm recorded according to (5), wherein, the base sequence that the asRNA of Ser1 is represented by sequence numbering 3 is constituted, the base sequence that the asRNA of Ser2 is represented by sequence numbering 7 is constituted, the base sequence that the asRNA of Ser3 is represented by sequence numbering 11 is constituted, and the base sequence that additionally FibH chain asRNA is represented by sequence numbering 14 is constituted.

(8) the gene recombinaton silkworm recorded any one of (5)～(7), above-mentioned expression vector is made up of 2 subunits.

(9) the gene recombinaton silkworm recorded according to (8), above-mentioned 2 subunits are present on different chromosomes.

(10) the gene recombinaton silkworm recorded any one of (5)～(9), has the expression of exogenous genes carrier that can express target allogenic gene.

(11) a kind of gene recombinaton silkworm manufacture method, it includes silkworm gives the operation of the expression of exogenous genes reinforcing agent of record any one of (1)～(4).

(12) method manufacturing the target peptide of allogenic gene coding in the silk glands of a kind of gene recombinaton silkworm recorded in (10).

This specification comprises Japan's patent application 2013-222138 description on the basis as the application priority and/or the content of accompanying drawing record.

Expression of exogenous genes reinforcing agent according to the present invention, by giving silkworm, in the silkworm of protein production system, it is possible to suppresses to become the sericin being mingled with protein or the synthesis of FibH chain.

Gene recombinaton silkworm according to the present invention, using the teaching of the invention it is possible to provide the repressed silkworm of synthesis of sericin and/or FibH chain. It addition, if having the gene recombinaton silkworm of the allogenic gene expressed by silk glands further, then production efficiency and the response rate of the target peptide of allogenic gene coding can be improved.

Peptide manufacture method according to the present invention, the gene recombinaton silkworm of the application of the invention is as protein production system, it is possible to manufacture the protein that added value is high in a large number and more at low cost.

Accompanying drawing explanation

Fig. 1 represents that the fibroin albumen expressed at silk glands and each position of silkworm constitutes the concept map before protein and discharge spun silk.

Fig. 2 indicates that the figure of the configuration example of the 1st subunit when the asRNA expression vector of the effective ingredient as expression of exogenous genes reinforcing agent of the present invention is made up of and the 2nd subunit 2 gene expression units. A～C is the configuration example of the 1st subunit. A configures a GAL4 gene part as the rear portion silk glands GAL4 expression vector of the DNA of encoding transcription regulatory factor under the silk glands promoter of rear portion. B is the some of the middle part silk glands GAL4 expression vector configuring GAL4 gene under the silk glands promoter of middle part. C is a part for & rear portion, the middle part silk glands GAL4 expression vector configuring GAL4 gene under middle part silk glands and the respective promoter of rear portion silk glands. D and E is the configuration example of the 2nd subunit. D is a part for the asRNA expression vector in the downstream of the UAS promoter of the target promoter as GAL4 with 1 asRNA coding DNA. E is a part for the asRNA expression vector with 2 series connection in the downstream of UAS promoter with asRNA coding DNA. In each subunit, the arrow of the 3 ' UTR that white edge represents represents transcriptional orientation.

Fig. 3 indicates that the figure of the composition of the 2nd subunit expressing Ser1asRNA that embodiment 1 grade uses. A is UAS-5 ' the Ser1asRNA expression vector of expression 5 ' Ser1asRNA. It addition, B is UAS-3 ' the Ser1asRNA expression vector of expression 3 ' Ser1asRNA. Herein, 5 ' Ser1asRNA using the 5 ' regions of the Ser1 that do not contain repetitive sequence region as target. It addition, 3 ' Ser1asRNA using the 3 ' regions of the Ser1 containing repetitive sequence region as target. The arrow of Ser1asRNA coding DNA represents transcriptional orientation (following, identical with this).

Fig. 4 indicates that the figure of the composition of the 2nd subunit expressing Ser1shRNA that embodiment 1 grade uses. A is UAS-5 ' the Ser1shRNA expression vector of expression 5 ' Ser1shRNA. It addition, B is UAS-3 ' the Ser1shRNA expression vector of expression 3 ' Ser1shRNA. Ser1shRNA is made up of Ser1 justice RNA (Ser1sRNA), connector (Actin intron) and Ser1asRNA.

Fig. 5 indicates that the figure of the composition of the 1st subunit that embodiment 1 grade uses, and represents the middle part silk glands GAL4 expression vector expressing GAL4 gene with middle part silk glands.

Fig. 6 indicates that have Fig. 3 and Fig. 4 each 2nd subunit represented and the figure of the expression inhibiting of the Ser1 gene of the gene recombinaton silkworm of the 1st subunit that Fig. 5 represents. In figure top record % be the average relative amount of each group of 3 systems (1,2,3) represented.

Fig. 7 indicates that have Fig. 3 and Fig. 4 each 2nd subunit represented and the figure of the SDS-PAGE of the cocoon protein of the gene recombinaton silkworm of the 1st subunit that Fig. 5 represents.

Fig. 8 indicates that the figure of the composition of the 2nd subunit expressing Fib-HasRNA that embodiment 2 grade uses. A is the UAS-Fib-H1asRNA expression vector expressing Fib-H1asRNA. It addition, B is the UAS-Fib-H2asRNA expression vector expressing Fib-H2asRNA. Fib-H1 and Fib-H2, the length as the repetitive sequence region of the FibH chain of target is different.

Fig. 9 indicates that the figure of the composition of the 1st subunit that embodiment 2 grade uses, and represents the rear portion silk glands GAL4 expression vector expressing GAL4 gene in the silk glands of rear portion.

Figure 10 indicates that have Fig. 8 the 2nd subunit represented and the figure of the expression inhibiting of the FibH chain gene of the gene recombinaton silkworm of the 1st subunit that Fig. 9 represents.

Figure 11 indicates that have Fig. 8 the 2nd subunit represented and the figure of the SDS-PAGE of the silk glands protein of the gene recombinaton silkworm of the 1st subunit that Fig. 9 represents.

Figure 12 indicates that the figure of the composition of the 2nd subunit (A) that embodiment 3 grade uses and the 1st subunit (B). With the 2nd subunit that A represents it is, downstream tandem in UAS promoter configures Ser1asRNA coding DNA and FibHasRNA coding DNA, can be expressed UAS-3 ' the Ser1-FibHasRNA expression vector of Ser1asRNA and FibHasRNA by the activity of UAS promoter simultaneously. Additionally B is, configure GAL4 gene respectively in the downstream of the Ser promoter (Ser-pro) as middle part silk glands promoter and the FibH promoter (Fib-Hpro) as rear portion silk glands promoter, utilize middle part and rear portion silk glands to express & rear portion, the middle part silk glands GAL4 expression vector of GAL4 gene simultaneously.

Figure 13 indicates that middle part silk glands GAL4 expression vector that & rear portion, the middle part silk glands GAL4 expression vector having Figure 12 B as the 1st subunit and representing, Fig. 5 represent or the rear portion silk glands GAL4 expression vector that Fig. 9 represents, and the figure of SDS-PAGE of silk glands protein of the gene recombinaton silkworm as Figure 12 A of the 2nd subunit UAS-3 ' Ser1-FibHasRNA expression vector represented.

Figure 14 indicates that the figure of the composition of expression of exogenous genes carrier. A is the basal expression carrier of the expression of exogenous genes carrier that embodiment 4 and 5 uses. B is the UAS-EGFP expression vector by 3xP3-EGFP, the marker gene of the expression vector of A being replaced with 3xP3-AmCyan and being used for embodiment.

Figure 15 indicates that the figure of the EGFP albumen quality of each gene recombinaton silkworm. Gene recombinaton silkworm used herein has a UAS-3 ' Ser1asRNA expression vector represented of the Fig. 3 as the 2nd subunit or Fig. 4 represents UAS-3 ' Ser1shRNA expression vector and the middle part silk glands GAL4 expression vector that the Fig. 5 as the 1st subunit represents and the UAS-EGFP expression vector that the Figure 14 as expression of exogenous genes carrier represents.

Figure 16 indicates that the figure of the EGFP albumen quality of each gene recombinaton silkworm. The UAS-EGFP expression vector that gene recombinaton silkworm used herein has Figure 12 UAS-3 ' Ser1-Fib-HasRNA expression vector as the 2nd subunit represented and & rear portion, middle part silk glands GAL4 expression vector and the Figure 14 as the 1st subunit represents.

Figure 17 indicates that the figure that the gelation expressing caused silk glands extracting solution of Ser1asRNA and Fib-HasRNA suppresses.

Detailed description of the invention

1. expression of exogenous genes reinforcing agent

1-1. summary

1st embodiment of the present invention is expression of exogenous genes reinforcing agent. The expression of exogenous genes reinforcing agent of the present invention, it is characterised in that containing the sericin gene will expressed in the silk glands of silkworm or FibH chain gene as the expression vector of the antisense RNA of target as effective ingredient. By silkworm being given the expression of exogenous genes reinforcing agent of present embodiment, it is possible to suppress the gene expression of sericin and/or FibH chain in silk glands.

1-2. defines

For the main terms used in this specification, make defined below.

" allogenic gene " is the foreignness gene being externally introduced via manual operation, is the gene can expressed in the silk glands of silkworm. As long as its source of the allogenic gene of this specification is the gene being externally introduced via manual operation, also include being so inserted into the gene existed under intrachromosomal state as the offspring of recombinant.

" target peptide " is by the peptide of above-mentioned foreignness gene code, refers to the peptide that should produce in the protein mass procution system of the silk glands employing silkworm. When this specification is designated as " peptide ", it it is the molecule that connected by amido link of the aminoacid of more than 2. Therefore, peptide includes oligopeptide and this kind of polypeptide of protein. Peptide, except deriving from individual gene or deriving from its genetic fragment, also includes deriving from a mosaic gene part for multiple genes being formed by connecting. It addition, the amino acid length of peptide, it does not have limit especially. Such as, total number of atnino acid can be 10～10000. The kind of the target peptide of this specification is not particularly limited, it is preferred to the protein that added value is high. Such as, this kind of peptide hormone of insulin, calcitonin, parathyroid hormone and growth hormone can be enumerated, epidermal growth factor (EGF), fibroblast growth factor (FGF), interleukin (IL), interferon (IFN), tumor necrosis factor α (TNF-α) and transforming growth factor β (TGF-β) this type cytokines, immunoglobulin, serum albumin, enzyme or collagen protein or their fragment (including chimeric peptide).

" expression of exogenous genes reinforcing agent " refers to the medicament of the expression strengthening the allogenic gene importing silkworm.

" Enhanced expressing " is that the expression instigating the allogenic gene in gene particularly this specification increases. In this specification, expression enhancing includes the increase of the absolute magnitude of the expression of (exogenous) gene and the increase of relative quantity. Herein, " increase of absolute magnitude " refer to (exogenous) gene expression itself increase. " increase of relative quantity " refer to the absolute magnitude of (exogenous) gene constant but by reduction of the expression of other genes etc. and the yield of target peptide increases relatively. Such as, can enumerate by suppressing FibH chain or sericin etc. to be mingled with the gene expression of protein in silk glands, thus compared with when not suppressing them, allogenic gene the yield of the target peptide encoded increases relatively.

" gene recombinaton silkworm " refers to genetic recombinants or its offspring of the silkworm with the recombinant DNA using gene recombination technology to make. The gene recombinaton silkworm of the present invention, particularly refers to the genetic recombinants or its offsprings that obtain the importing of host silkworm as the asRNA expression vector of the effective ingredient of the expression of exogenous genes reinforcing agent of present embodiment more.

The pipe of the salivary gland change that " silk glands " is able to the insecticide of discharge spun silk, namely spun silk worm has, it has production, accumulates and secrete the function of aqueous silk. Silk glands exists in the way of pair of right and left primarily along the digestive tube of the larva of spun silk worm, each silk glands is made up of 3 regions at anterior, middle part and rear portion silk glands, at middle part silk glands synthesis secretion sericin, and at rear portion silk glands synthesis secretion FibH chain, FibL chain and p25.

" sericin (Ser) " is as noted above is water soluble gelatine sample protein (Fig. 1) covering composition as spun silk. At middle part, silk glands is synthesized, and secretes to middle part silk glands inner chamber. In sericin known have Ser1, Ser2, Ser3 by multiple molecular speciess of each gene chemical synthesis, but when this specification is only recited as " sericin ", be the general name including all molecular speciess.

One of 3 main protein of fibroin albumen of " fibroin albumen H chain (FibH) " fibre composition being composed as spun silk as noted above (Fig. 1). At rear portion, silk glands is synthesized, and after collectively forming SFEU complex with fibroin albumen L chain and p25, secretion is to the silk glands inner chamber of rear portion.

1-3. is constituted

The expression of exogenous genes reinforcing agent of present embodiment contains antisense RNA expression vector as essential component, in addition, contains carrying body as required as selection component. Hereinafter, each composition is illustrated.

1-3-1. antisense RNA expression vector

" antisense RNA expression vector " (in this specification, is usually designated as " asRNA expression vector "), is the effective ingredient of the expression of exogenous genes reinforcing agent of present embodiment. The parent nucleus of asRNA expression vector can utilize various expression vector. Such as, can enumerate plasmid or rod granule (Bacmid) this kind of can independently replicate expression vector, viral vector or can the expression vector of homologous recombination or non-homogeneous restructuring or the chromosomal part in being inserted into the chromosome of host in chromosome. It addition, the shuttle vector that asRNA expression vector can also be able in other antibacterials such as escherichia coli to be replicated.

The constitution element of A.asRNA expression vector

AsRNA expression vector, is constituted in the way of expressing in the silk glands of silkworm for the asRNA of target gene. Promoter required for containing the expression of the DNA and this asRNA that encode at least one asRNA in the constitution element of expression vector is as necessary constitution element. It addition, above-mentioned asRNA other constitution elements expressed can be contributed to as required. Herein, other constitution elements, for instance the inverted terminal repeat sequence etc. of terminator, marker gene, enhancer, insulator and transposon can be enumerated. And, when asRNA expression vector is made up of 2 unit of the 1st subunit described later and the 2nd subunit, comprise the DNA of encoding transcription regulatory factor and the target promoter of this transcription regulaton factor as necessary constitution element. Although it addition, be not the constitution element of the expression directly participating in asRNA, but this carrier can also contain allogenic gene. Hereinafter, the composition of each element is illustrated.

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

The DNA of encoding antisense RNA is (in this specification, usually referred to as " asRNA coding DNA "), encode sericin 1～3 specific expressed for the middle part silk glands silkworm or the asRNA suppressing its expression at the specific expressed fibroin albumen H chain gene of rear portion silk glands as target.

Using sericin 1 (Ser1) gene asRNA (Ser1asRNA) as target, in the full length amino acid sequence of silkworm Ser1 protein sequence numbering 1 represented, 628～1054 repetitive sequence regions existed are as target. The base sequence of Ser1asRNA contains the complementary series of the base sequence of the coding repetitive of at least one contained by this repetitive sequence region. The repetitive in the repetitive sequence region of Ser1, sequence numbering 2 the aminoacid sequence SX represented₁SNTDX₂SX₁X₃X₄X₅GSX₆TSGGX₁STYGYSSX₇X₈RX₉GSVSX₂The derived sequence of TG and its phase place of change is constituted. Herein, X₁Represent T or S, X₂Represent S or A, X₃Represent K or D, X₄Represent S, N or L, X₅Represent A or T, X₆Represent R or S, X₇Represent R, S or D, X₈Represent H, S or N, additionally X₉Represent G or D.

In this specification, " derived sequence of change phase place " refers to the sequence of the beginning amino acid position displacement making repetitive in repetitive sequence region. Such as, as the derived sequence of phase place of the repetitive changing the expression of above-mentioned sequence numbering 2, can enumerate from by beginning amino acid position to 2 amino acid whose SNT of C-terminal side displacement, at GSX₁The sequence etc. terminated. Usual repetitive in repetitive sequence region in continuously repeat appearance, even if the entity of repetitive sequence is also constant when therefore the initial amino acid of repetitive is with global displacement. Therefore, the derived sequence of the phase place of repetitive is changed, it is believed that the repetitive substantially the same with the concrete repetitive represented of sequence numbering. As the concrete example of the base sequence of Ser1asRNA, the base sequence that sequence numbering 3 represents can be enumerated. Therefore, the DNA sequence of the sequence that coded sequence numbering 3 represents, it is possible to become one of Ser1asRNA coding DNA.

Using sericin 2 (Ser2) gene as the asRNA (Ser2asRNA) of target be in the full length amino acid sequence of the silkworm Ser2 protein that sequence numbering 4 is represented the repetitive sequence region of 41～117 and 685～1359 existence as target. The base sequence of Ser2asRNA contains the complementary series of the base sequence of any one at least one repetitive contained by repetitive sequence region encoding these 2 regions. The repetitive in the repetitive sequence region of Ser2 is respectively present in above-mentioned 2 regions, 41～117 the 1st repetitives existed, sequence numbering 5 the aminoacid sequence KX represented₁EX₂X₃KX₄X₅X₆GX₄Constitute with the derived sequence changing its phase place. Herein, X₁Represent F, L or V, X₂Represent N or A, X₃Represent L, I or A, X₄Represent E or D, X₅Represent N or K, X₆Represent V or A. It addition, the aminoacid sequence X that 685～1359 the 2nd repetitives existed are represented by sequence numbering 6₁SX₂SX₃X₄DX₅X₆KX₇X₈X₉X₁₀X₁₁Constitute with the derived sequence changing its phase place. Herein, X₁Represent G, R or S, X₂Represent S or P, X₃Represent D, H or Y, X₄Represent K or R, X₅Represent S or T, X₆Represent E or D, X₇Represent A, V or T, X₈Represent K or F, X₉Represent P, H or D, X₁₀Represent N or K, additionally X₁₁Represent D, G or N. As the concrete example of the base sequence of Ser2asRNA, the base sequence that sequence numbering 7 represents can be enumerated. Therefore, the DNA sequence of the sequence that coded sequence numbering 7 represents becomes one of Ser2asRNA coding DNA.

Using sericin 3 (Ser3) gene as the asRNA (Ser3asRNA) of target be in the aminoacid sequence of the silkworm Ser3 protein that sequence numbering 8 is represented the repetitive sequence region of 141～978 and 1031～1178 existence as target. The base sequence of Ser3asRNA contains the complementary series of the base sequence of any one at least one repetitive contained by repetitive sequence region encoding these 2 regions. The repetitive in the repetitive sequence region of Ser3 is respectively present in above-mentioned 2 regions, the aminoacid sequence SDDSSGATKGNSSKSSSSSQGX that 141～978 the 1st repetitives existed are represented by sequence numbering 9₁SASSSSSX₂EX₃SSQSX₄SNSSNNSKSSSQSSSX₅X₆NSSGSKGSGSEESSNGGSGSGRX₇GSX₈GX₉X₁₀The derived sequence of DED and its phase place of change is constituted. Herein, X₁Represent Q or K, X₂Represent D or N, X₃Represent K or N, X₄Represent S or N, X₅Represent S or G, X₆Represent Q, N or K, X₇Represent T or N, X₈Represent A or V, X₉Represent G or E, additionally X₁₀Represent T or S. It addition, the aminoacid sequence X that 1031～1178 the 2nd repetitives existed are represented by sequence numbering 10₁SX₂SX₃QAX₄Constitute with the derived sequence changing its phase place. Herein, X₁Represent S or N, X₂Represent R or S, X₃Represent Q or K, additionally X₄Represent Q or H. As the concrete example of the base sequence of Ser3asRNA, the base sequence that sequence numbering 11 represents can be enumerated. Therefore, the DNA sequence of the sequence that coded sequence numbering 11 represents can become one of Ser1asRNA coding DNA.

Being that in the aminoacid sequence of silkworm FibH protein sequence numbering 12 represented, 152～5203 the repetitive sequence regions existed are as target as the asRNA (Fib-HasRNA) of target using fibroin albumen H (FibH) chain gene, the base sequence of Fib-HasRNA contains the complementary series of the base sequence encoding this at least one repetitive contained by repetitive sequence region. The aminoacid sequence GX that the repetitive in the repetitive sequence region of FibH is represented by sequence numbering 13₁GX₁GX₂Constitute with the derived sequence changing its phase place. Herein, X₁Represent A, V or Y, additionally X₂Represent S or Y. As the concrete example of the base sequence of FibHasRNA, the base sequence that sequence numbering 14 represents can be enumerated. Therefore, the DNA sequence of the sequence that coded sequence numbering 14 represents, it is possible to become one of FibHasRNA coding DNA.

(2) promoter

Promoter acts on the asRNA coding DNA of asRNA expression vector, is the necessary constitution element expressing above-mentioned asRNA.

The kind of promoter contained by asRNA expression vector is different because the target gene of asRNA is different. Such as, when the gene of the Ser1～Ser3 expressed by middle part silk glands is as target gene, using can in the promoter of middle part silk glands work. In such promoter, for instance outside a good appetite suddenly appearing in a serious disease portion silk glands specificity promoter, also can enumerate generally can express overexpression type promoter, composition active form promoter or period specific activity type promoter or induced expression type promoter etc. It is preferably middle part silk glands specificity promoter. Silk glands is special and the promoter of the gene of the protein of great expression to be wherein particularly preferably coding middle part. Specifically, for instance, the gene promoter (in this specification, being called " Ser1 promoter ", " Ser2 promoter " and " Ser3 promoter ") of Ser1～Ser3 can be enumerated. On the other hand, when the FibH chain gene expressed by rear portion silk glands is as target gene, using can in the promoter of rear portion silk glands work. In such promoter, for instance, except the silk glands specificity promoter of rear portion, can enumerate generally can express overexpression type promoter, composition active form promoter or period specific activity type promoter or induced expression type promoter etc. It is preferably rear portion silk glands specificity promoter. Wherein it is particularly preferably that coding rear portion silk glands is special and the promoter of the gene of the protein of great expression. Specifically, for instance the gene promoter (in this specification, being called " FibH promoter ", " FibL promoter " or " p25 promoter ") of FibH, FibL or p25 of constituting protein as fibroin albumen can be enumerated.

As long as the source organism kind of promoter can work in the cell of the silkworm of importing asRNA expression vector, it does not have limits especially. For example, it is preferable to be the silk glands specificity promoter deriving from spun silk worm. Generally, middle part silk glands specificity promoter or rear portion silk glands specificity promoter base sequence due between spun silk worm evolution camber guard, even if thus be accordingly used in the promoter of the expression of asRNA to derive from the spun silk worm different from silkworm, also can function (SezutsuH. in the cell of silkworm, etal., 2009, JournalofInsectBiotechnologyandSericology, 78:1-10). The biological species of preferred promoter is belong to the kind of identical Lepidoptera (Lepidoptera) on taxonomy with the silkworm of the host becoming asRNA expression vector, it is more preferably the kind of the Bombycidae (Bombycidae) belonging to identical, more preferably such as belong to the kind of the so identical genus of Bombyx mandarina (Bombyxmandarina), it is most preferred that for silkworm.

As concrete example, it is possible to utilize following promoter. In the silk glands specificity promoter of middle part, it is possible to the Ser1 promoter deriving from silkworm that utilizes the base sequence represented containing sequence numbering 15, the Ser2 promoter deriving from silkworm of the base sequence represented containing sequence numbering 16 and the Ser3 promoter etc. deriving from silkworm of base sequence containing sequence numbering 17 expression. Additionally, in the silk glands specificity promoter of rear portion, it is possible to the FibH promoter deriving from silkworm that utilizes the base sequence represented containing sequence numbering 18, the FibH promoter deriving from Antherea pernyi Guerin-Meneville of the base sequence represented containing sequence numbering 19, the FibL promoter deriving from silkworm of base sequence represented containing sequence numbering 20, the FibL promoter deriving from Antherea pernyi Guerin-Meneville of base sequence containing sequence numbering 21 expression and the p25 promoter etc. deriving from silkworm of base sequence containing sequence numbering 22 expression.

(3) terminator

Terminator is made up of the base sequence transcribed that can terminate asRNA in the rear portion silk glands cell of the host of importing asRNA expression vector.

(4) marker gene

Marker gene is the gene that coding is also referred to as the labelled protein selecting label. Labelled protein refers to polypeptide with presence or absence of the expression that can interpolate that marker gene based on its activity. Therefore, asRNA expression vector is by containing marker gene, it is possible to the activity based on labelled protein easily determines the gene recombinaton silkworm with asRNA expression vector. Refer to " testing result based on activity " " based on activity " herein. The detection of activity, it is possible to be the activity of directly detection labelled protein itself, it is also possible to be via this metabolite indirect detection of pigment generated by the activity of labelled protein. Detection can be any one of the photoreceptor apoptosis (including utilizing the detection of vision, sense of touch, olfactory sensation, audition, the sense of taste) of chemical detection (including the detection of enzyme reaction), physical detection (including the detection of action analysis) or tester.

As long as the kind of labelled protein can detect its activity by this field known method, it does not have limits especially. It is preferably labelled protein during detection, the aggressive of gene recombinaton silkworm is low. Such as, the protein etc. of fluorescence protein, pigment synthesis protein, photoprotein, external secretion protein, control formalness can be enumerated. Fluorescence protein, pigment synthesis protein, photoprotein and external secretion protein, due under given conditions can vision-based detection, therefore that the aggressive of gene recombinaton silkworm is non-normally low, it is judged that with select easily, so particularly preferably.

Above-mentioned fluorescence protein sends the protein of the fluorescence of specific wavelength when referring to the exciting light that gene recombinaton silkworm is irradiated specific wavelength. It can be any one of natural type and non-natural type. It addition, excitation wavelength, wavelength of fluorescence are also without special restriction. Specifically, for instance, CFP, AmCyan, RFP, DsRed (including this analog derivative of DsRedmonomer, DsRed2), YFP, GFP (including the derivants such as EGFP, EYFP) etc. can be enumerated.

Above-mentioned pigment synthesis protein is the biosynthetic protein participating in pigment, is generally enzyme. So-called " pigment " low molecular compound or the peptide being able to transformant gives pigment herein, its kind is not limit. It is preferably the pigment as individual exterior color performance. Such as, black prime system pigment (including dopamine melanin), wink prime system pigment or pteridine system pigment can be enumerated.

Above-mentioned photoprotein refer to need not exciting light can be luminous substrate protein white matter or the luminous enzyme of this substrate protein white matter of catalysis. Such as, aequorin, luciferase as enzyme can be enumerated.

This specification peripheral secretory protein is to secrete to extracellular or external protein, belongs to exocrinosity enzyme etc. In exocrinosity enzyme, including contribute to this kind of medicament of blasticidin S decompose or inactivation and give the enzyme of host's drug resistance, in addition also include digestive enzyme.

Marker gene configures with the state that can express in the downstream of above-mentioned promoter in asRNA expression vector. The promoter used can be identical with asRNA coding DNA, it is also possible to different.

(5) insulator

Insulator is the sequence transcribed of the gene that stability contorting is clamped by this sequence under the chromosomal chromatin being not subjected to surrounding affects. Such as, the gypsy sequence etc. of the cHS4 sequence of chicken, fruit bat can be enumerated.

(6) inverted terminal repeat sequence of transposon

Inverted terminal repeat sequence (the Invertedterminalrepeatsequence) (HandlerAM.etal. of transposon, 1998, Proc.Natl.Acad.Sci.U.S.A.95:7520-5) can be contained when asRNA expression vector is the asRNA expression vector that can recombinate. Inverted terminal repeat sequence is configured at the upstream and downstream of asRNA expression vector. As transposon, it is possible to and use piggyBac, mariner, minos etc. (Shimizu, K.etal., 2000, InsectMol.Biol., 9,277-281; WangW.etal., 2000, InsectMolBiol9 (2): 145-55).

(7) DNA of encoding transcription regulatory factor

" DNA of encoding transcription regulatory factor " is the essential elements of the 1st subunit described later, refers to the gene of transcription regulaton factor. In this specification, so-called " transcription regulaton factor " referring to is combined with target promoter described later, it is possible to activate the rho factor of this target promoter. Such as, the GAL4 protein of the galactose metabolism activation of protein as yeast can be enumerated and as the tTA and mutant etc. thereof of tetracycline controlling transcriptional activators.

(8) the target promoter of transcription regulaton factor

" the target promoter of transcription regulaton factor " is the essential elements of the 2nd subunit described later, refers to by being combined by the transcription regulaton factor of the 1st subunit coding, can activate the promoter of certain gene expression at the control. Above-mentioned transcription regulaton factor and target promoter thereof, have corresponding relation with above-mentioned transcription regulaton factor, generally, if it is determined that transcription regulaton factor, then also necessarily is able to determine its target promoter. Such as, when transcription regulaton factor is GAL4 protein, use UAS (UpstreamActivatingSequence).

(9) allogenic gene

Allogenic gene, described in as defined above, is the foreignness gene can expressed in silk glands, encoding target peptide. The asRNA that the original function of the asRNA expression vector contained by expression of exogenous genes reinforcing agent of present embodiment is the expression making the target genes such as suppression Ser1～Ser3, FibH chain expresses. Therefore, the allogenic gene of encoding target peptide, with asRNA expression vector be constructed without direct relation. But, the purpose of asRNA is the expression by suppressing above-mentioned target gene, indirectly strengthens the expression of allogenic gene, efficient productive target peptide. That is, allogenic gene is to play object necessary on the basis of its effect at the expression of exogenous genes reinforcing agent of present embodiment. Therefore, the host insect of the expression of exogenous genes reinforcing agent of present embodiment is given when not there is the allogenic gene can expressed in silk glands, it is impossible to fully realize the purpose of the present invention. The allogenic gene of encoding target peptide, generally, is contained in other expression vector (expression of exogenous genes carrier) described later, imports identical host insect independently mutually with asRNA expression vector. But it is also possible to be contained in asRNA expression vector when the allogenic gene as this object can be expressed. When asRNA expression vector contains allogenic gene, in order to allogenic gene can be expressed in silk glands, above-mentioned middle part silk glands specific promoter and/or rear portion silk glands specific promoter can be configured at its upstream.

The unit of B.asRNA expression vector is constituted

AsRNA expression vector includes the situation being made up of 1 gene expression units and the situation being made up of 2 gene expression units. Hereinafter each situation is illustrated.

(1) situation being made up of 1 gene expression units

When asRNA expression vector is made up of 1 gene expression units, as long as silkworm being given the expression of exogenous genes reinforcing agent containing this asRNA expression vector, it becomes possible at silkworm cell inner expression purpose asRNA. Whole constitution elements necessary in expressing asRNA are contained in 1 unit by this kind of asRNA expression vector. That is, at least 11 group of expression system being made up of the promoter as necessary constitution element and the asRNA coding DNA in the functional combination in the downstream of this promoter is comprised. This 1 group of expression system can be the system under 1 promoter controls containing 1 asRNA coding DNA, it is also possible to be the system containing more than 2 asRNA coding DNAs.

(2) situation (Fig. 2) being made up of 2 gene expression units

When asRNA expression vector is made up of the 1st subunit and 2 gene expression units of the 2nd subunit, constitution element necessary in expressing asRNA is individually separated being present in 2 unit. Therefore, as long as these 2 exist in bombyx mori cell and just as 1 asRNA expression vector function, can express purpose asRNA. Each subunit has following composition.

1st subunit contains promoter and forms at the DNA encoding above-mentioned transcription regulaton factor of the functional combination in the downstream of this promoter. Fig. 2 A～C illustrates the 1st subunit. The promoter used in 1st subunit, in the same manner as the promoter of the expression with above-mentioned control asRNA coding DNA, being used in silkworm silk glands can the promoter of function. Such as, can enumerating that FibH, FibL or p25 etc. as shown in Fig. 2 A rear portion silk glands promoter represented are special at rear portion silk glands and the gene promoter of the protein of a large amount of synthesis, sericin protein as shown in Fig. 2 B middle part silk glands promoter represented etc. is at the gene promoter of the protein of and a large amount of synthesis special at middle part silk glands. As shown in Figure 2 C, in the 1st subunit, there are the many groups of groups being made up of the DNA of promoter and encoding transcription regulatory factor, it is possible to each promoter is set as rear portion silk glands promoter and the so different combination of middle part silk glands promoter. Should illustrate, in Fig. 2 A～C, as the DNA of encoding transcription regulatory factor, illustrate GAL4 gene.

2nd subunit contains the target promoter of the above-mentioned transcription regulaton factor by above-mentioned 1st subunit coding and the asRNA coding DNA of functional combination forms with the downstream of this target promoter. Fig. 2 D and E represents an example of the 2nd subunit. In Fig. 2 D, as the target promoter of GAL4, illustrate UAS. It addition, the 2nd subunit, except containing the system of 1 asRNA coding DNA as shown in Figure 2 D under 1 target promoter controls, it is also possible to be the system containing more than 2 asRNA coding DNAs shown in Figure 2 E under 1 target promoter controls. Should illustrate, when asRNA expression vector contains the allogenic gene of encoding target peptide, it is preferable that be contained in the 2nd subunit.

The asRNA expression vector of this composition, 1 group of function with the 1st and the 2nd subunit as noted above. Expressed the activation of promoter by silk glands, the 1st subunit the target promoter of transcription regulaton factor activation the 2nd subunit expressed, thus expressing purpose asRNA. It addition, the 2nd subunit can be made up of more than 2 subunits containing identical or different asRNA coding DNA. Now, 1 the 1st subunit the transcription regulaton factor expressed, by activating the target promoter of multiple 2nd subunit, it is possible to expresses by the asRNA of each the 2nd subunit coding.

The asRNA expression vector of this composition, via the expression amplification of the asRNA coding DNA that can be made the 2nd subunit by the transcription regulaton factor of the 1st subunit coding. Therefore, when the ability to express that the silk glands of the 1st subunit expresses promoter is not high, it is particularly preferred to.

The asRNA expression vector of this composition, as long as only by the asRNA encoding DNA region of the 2nd subunit with expression cassette and the exchange of other asRNA encoding DNA region, existing for the situation that the 1st subunit can be used in conjunction with. That is, the 1st subunit also is able to utilize existing expression vector or import its gene recombinaton man mori system. Therefore, the asRNA expression vector being made up of 2 gene expression units, manufacture express unit, the one-tenth present aspect of gene recombinaton silkworm and work in be also convenient.

Should illustrate, when asRNA expression vector is made up of the 1st subunit and 2 subunits of the 2nd subunit, the expression of exogenous genes reinforcing agent of present embodiment, in principle by constituting as the form of 2 doses 1 group of the 2nd expression of exogenous genes reinforcing agent of effective ingredient containing the 1st subunit the 1st expression of exogenous genes reinforcing agent as effective ingredient with containing the 2nd subunit. As noted above, 1st subunit is owing to being used in conjunction with, therefore the 1st expression of exogenous genes reinforcing agent and the 2nd expression of exogenous genes reinforcing agent it is not absolutely required to be the correspondence of 1 pair 1, it also is able to obtain the 1st expression of exogenous genes reinforcing agent as common agent, the only composition of the combination of various 2 doses 1 group of exchange the 2nd expression of exogenous genes reinforcing agent. Additionally, give the host insect of the expression of exogenous genes reinforcing agent of present embodiment, if having had the gene recombinaton insecticide of the 1st subunit in chromosome, then the expression of exogenous genes reinforcing agent of present embodiment can only be constituted as the 2nd expression of exogenous genes reinforcing agent of effective ingredient by using the 2nd subunit corresponding with the 1st subunit.

1-3-2. carrying body

The expression of exogenous genes reinforcing agent of the present invention, can contain acceptable carrying body as required. " acceptable carrying body " can enumerate normally used solvent, adjuvant etc. in entomology field.

In solvent, for instance, water or aqueous solution can be enumerated or for the acceptable organic solvent of silkworm. As aqueous solution, for instance, buffer (phosphate buffer, sodium acetate buffer etc.), normal saline solution, isotonic solution can be enumerated. Generally, water or aqueous solution are used.

In adjuvant, for instance, assistant carrier can be enumerated. Assistant carrier, for instance, containing the DNA of coding transposon transferring enzyme. When having the inverted terminal repeat sequence of transposon as the asRNA expression vector of the effective ingredient of expression of exogenous genes reinforcing agent, assistant carrier is by producing transposon transferring enzyme, it is possible to realize in the chromosome that asRNA expression vector inserts host insect. As assistant carrier, if host insect is the situation of silkworm, for instance, pHA3PIG can be enumerated. As other adjuvant can enumerate glucose, D-Sorbitol, D-MANNOSE, PEARLITOL 25C, sodium chloride, other can also be the nonionic surfactant of low concentration, polyoxyethylene sorbitan fatty acid ester class etc.

2. gene recombinaton silkworm

2-1. summary

Second embodiment of the present invention is gene recombinaton silkworm. The gene recombinaton silkworm of present embodiment, it is characterised in that there is the asRNA expression vector that above-mentioned 1st embodiment is recorded. When present embodiment gene recombinaton silkworm has expression of exogenous genes carrier further, asRNA expression vector the asRNA expressed suppresses to become the expression of the endogenous gene such as the sericin of its target, FibH chain. Efficiently produce, in silk fabric gland, the target peptide encoded by allogenic gene thereby, it is possible to be in.

2-2. is constituted

The gene recombinaton silkworm of the present invention has the asRNA expression vector that above-mentioned 1st embodiment is recorded. About the composition of asRNA expression vector, the description thereof will be omitted due to identical therefore with the asRNA expression vector that the 1st embodiment is recorded, and the distinctive composition of gene recombinaton silkworm of present embodiment is illustrated herein.

In gene recombinaton silkworm, the asRNA expression vector that the 1st embodiment is recorded, it is possible to be temporarily present in bombyx mori cell, sustainable existence can also be stablized importing the state etc. in chromosome in addition. Generally, it is preferred to stable and sustainable existence.

Gene recombinaton silkworm can have the asRNA expression vector of more than different 2 that the 1st embodiment is recorded. Such as, gene recombinaton silkworm, it is possible to there is both the expression vector being made up of 1 gene expression units and expression vector of being made up of 2 gene expression units. Now, each asRNA expression vector can contain identical or different asRNA coding DNA.

Gene expression units is as noted above to be made up of the 1st subunit and the 2nd subunit 2, and when each existing on the chromosome of silkworm, each subunit can exist on phase homologous chromosomes, it is also possible to exist on different chromosomes. When each subunit exists on different chromosomes, by the systematic mating of the system of the gene recombinaton silkworm by only having the 1st subunit (being preferably homozygote) and the gene recombinaton silkworm only with the 2nd subunit (being preferably homozygote), F1 can be readily obtained the gene recombinaton silkworm of the present invention with the 1st subunit and the 2nd subunit. Now, the system of the above-mentioned gene recombinaton silkworm only with the 1st subunit, as shown in the 1st embodiment records, it is possible to be provided commonly for the copulation of system with the various gene recombinaton silkworms only with the 2nd subunit.

On the other hand, when the 1st subunit and the 2nd subunit exist on phase homologous chromosomes, separated from one another in order to not cause because recombinating in succeeding generations, it is preferable that the distance between subunit is near, interrelated.

The gene recombinaton silkworm of present embodiment, it is not absolutely required to the allogenic gene containing encoding target peptide. As noted above, this is because have been provided with by have asRNA expression vector suppress target gene expression, as the function of gene recombinaton silkworm of present embodiment. But, if only having the composition of asRNA expression vector, it is impossible to the silk fabric gland that realizes being in improves this purpose of the present invention of production efficiency of target peptide encoded by allogenic gene. In order to silk fabric gland of being in improves the production efficiency of target peptide, the gene recombinaton silkworm of present embodiment needs the existence of the allogenic gene as the object that should play this function. Therefore, using the gene recombinaton silkworm of present embodiment as protein mass procution system use time, except asRNA expression vector, in addition it is also necessary to have the expression system (expression of exogenous genes carrier) of the allogenic gene of encoding target peptide further. Expression of exogenous genes carrier, as required, it is possible to prepare in the way of the gene recombinaton silkworm of present embodiment is possessed in advance. Should illustrating, when asRNA expression vector contains the allogenic gene of encoding target peptide, the gene recombinaton silkworm of present embodiment also is able to realize the purpose of the present invention by having asRNA expression vector simultaneously.

2-3. manufacture method

The method that the manufacture method of the gene recombinaton silkworm of present embodiment is able to make the recombinant silkworm that the expression vector recorded by above-mentioned 1st embodiment expresses purpose asRNA, it is also possible to adopt arbitrary method, it does not have limit especially. As the method making such recombinant silkworm, such as, can enumerate the expression of exogenous genes reinforcing agent of the above-mentioned 1st embodiment method to giving as the silkworm of host, will there is the 1st subunit of asRNA expression vector that the 1st embodiment records and the method that the gene recombinaton silkworm of the 2nd subunit carries out copulation on different chromosomes.

The method that silkworm is given by the expression of exogenous genes reinforcing agent of above-mentioned 1st embodiment, it is possible to undertaken by the asRNA expression vector of the effective ingredient as expression of exogenous genes reinforcing agent can be imported the method well known in the art of silkworm. Such as, when silkworm egg being given by the expression of exogenous genes reinforcing agent of the 1st embodiment, it is possible to utilize the method (TamuraT.etal. of the Tamura etc. that expression vector is imported silkworm, 2000, NatureBiotechnology, 18,81-84). Specifically, becoming the mode of suitable concentration according to the asRNA expression vector comprised and expression of exogenous genes reinforcing agent utilizes water, buffer equal solvent dissolve or dilution, preparation gives solution. Herein, asRNA expression vector is when the two ends of asRNA coding DNA have the inverted terminal repeat sequence of transposon, it is possible to adds the assistant carrier of the DNA with coding transposon transferring enzyme in giving solution, is injected to the early development ovum of silkworm. The silkworm now used, it is possible to be wild type silkworm, it is also possible to be with the gene recombinaton silkworm with expression of exogenous genes carrier of silkworm silk glands productive target peptide. It addition, when expression of exogenous genes reinforcing agent contains assistant carrier, it is possible to by the above-mentioned solution direct injection that gives to silkworm development initial stage ovum. Then, by based on selecting label to select transformant, it is possible to obtain genes of interest recombinant silkworm. In the gene recombinaton silkworm obtained in this way, the asRNA expression vector in expression of exogenous genes reinforcing agent is assembled in chromosome via the inverted terminal repeat sequence of transposon. As required this gene recombinaton silkworm can be carried out sib mating or inbreeding, obtain inserting the homozygote of the expression vector in chromosome.

On the other hand, the 1st subunit of expression vector and the gene recombinaton silkworm of the 2nd subunit on different chromosomes respectively with the 1st embodiment record are carried out copulation and can also carry out by means commonly known in the art to the method making the gene recombinaton silkworm of the present invention. Such as, first, each silkworm is given respectively by the 1st expression of exogenous genes reinforcing agent containing each subunit and the 2nd expression of exogenous genes reinforcing agent. Administration way, as described above can method by expression vector imports silkworm, for instance can be undertaken by the method for Tamura etc. Now, for instance, the gene recombinaton silkworm on chromosome with the 1st subunit has been established, it is possible to utilize at that time, it is possible to only the 2nd expression of exogenous genes reinforcing agent containing 2nd subunit corresponding with the 1st subunit is imported silkworm. Such as, the gene recombinaton silkworm of the 1st subunit in chromosome with the gene of the downstream configuration codes GAL4 protein in this kind of middle part of sericin promoter silk glands promoter has been established, can utilize at that time, only can give silkworm by the 2nd expression of exogenous genes reinforcing agent, making the gene recombinaton silkworm on chromosome with the 2nd subunit, described 2nd expression of exogenous genes reinforcing agent contains the 2nd subunit of the downstream configuration purpose asRNA coding DNA of the UAS promoter at the target sequence as GAL4 protein as effective ingredient. The gene recombinaton silkworm with each subunit obtained carries out sib mating or inbreeding, is preferably made as homozygote in advance about each subunit. It follows that the gene recombinaton silkworm with each subunit is carried out copulation, by selecting the F1 with 2 subunits individual, it is possible to obtain genes of interest recombinant silkworm. Choice is based on the 1st and the 2nd respective choice label of subunit and carries out.

After making the gene recombinaton silkworm of present embodiment, expression of exogenous genes carrier can also be imported as required further. Now, by the method for the gene recombinaton silkworm of expression of exogenous genes vector introduction present embodiment, it is possible to use method similar to the above carries out.

3. peptide manufacture method

3-1. summary

3rd embodiment of the present invention is peptide manufacture method. Manufacture method according to present embodiment, at the silk glands of gene recombinaton silkworm, it is possible to the target peptide of high efficiency manufacture allogenic gene coding when suppressing sericin, FibH chain etc. to be mingled with protein expression.

3-2. material

The peptide manufacture method of present embodiment uses gene recombinaton silkworm as the mass procution system of protein. The gene recombinaton silkworm that present embodiment uses is the gene recombinaton silkworm of asRNA expression vector and the expression of exogenous genes carrier with the 1st embodiment record. That is, in other words it is the gene recombinaton silkworm of above-mentioned 2nd embodiment with expression of exogenous genes carrier.

The expression of exogenous genes carrier that the gene recombinaton silkworm that present embodiment uses has, comprises the allogenic gene of the target peptide that coding should manufacture in the peptide manufacture method of present embodiment.

3-3. manufacture method

The manufacture method of the present invention, including raising operation and recovery process. Hereinafter, each operation is illustrated.

(1) operation is raised

" raising operation " is the operation raising gene recombinaton silkworm. Method for breeding about gene recombinaton silkworm, it is possible to raise according to the breeding technology of silkworm well known in the art. For example, it is possible to reference to " silkworm egg introduction; Brilliant idea husband work, whole nation silkworm egg association periodical ". Feedstuff can use the natural leaf of this kind of food grass seeds of leaf of Morus (Morus), it is possible to use SilkMateL4M or original silkworm egg 1-3 uses (Japanese agriculture industry) this kind of man-made feeds age. Occur from suppressing disease, it is possible to carry out providing bait with stable quality and quantity, and can the angle of aseptic raising set out as required, it is preferred to man-made feeds. Hereinafter, for the simple method for breeding of gene recombinaton silkworm, illustrate as an example.

Children silkworm moves to the operation that silkworm rearing bed carries out raising be the female ovum laid eggs of the homologous gene recombinant silkworm utilizing suitable number of elements (such as, 4～10) and carry out. The larva of hatching is transferred in the container being covered with anti-dry paper (paraffin processing paper) of silkworm rearing bed from silkworm seed paper, the man-made feeds such as SilkMate is placed in anti-dry paper and lifts up for bait. The exchange of bait, respectively carries out 1 time in 1～2 age, carries out 1～3 time in 3 ages in principle. Old bait eat surplus many time, in order to prevent corruption to be removed. Raising when the healthy and strong silkworm larva in 4～5 ages, is transferred to tun, suitably adjusts the number of elements of each container. According to the state in humidity, container, container can also cover the lid of anti-dry paper, acrylic resin or steel wire. Raising temperature, runs through and raises at 25～28 DEG C full age.

(2) recovery process

" recovery process " is to reclaim the operation of target peptide that the larva of gene recombinaton silkworm is accumulated in silk glands.

The gene recombinaton silkworm that present embodiment uses, mainly starts to express allogenic gene from the last later stage in age of larva, and the target peptide of its coding is at silk glands intracellular accumulation. The method reclaiming target peptide from this gene recombinaton silkworm does not limit. Such as, can enumerate and from the end later stage in age to prepupal period from polypide, silk glands extracted, the method directly reclaiming target peptide. Concrete method can realize by means commonly known in the art. Such as, after silkworm before will the weaving silk of (5 age) the 6th day in age of end is anaesthetized on ice, being cut by dorsal part utilizes pin etc. to extract (with reference to gloomy quiet volume in the way of not injuring silk glands, the neontology utilizing silkworm is tested, three province's halls, 1970, pp.249-255). The silk glands of extraction is such as slowly vibrated at the temperature of 0～10 DEG C, preferably 0～5 DEG C in said extracted buffer, it is possible to target peptide is dissolved in buffer. If target peptide is not heat sensitivity peptide, it is possible to carry out at the temperature of 10～40 DEG C. Then, by centrifugal or filtration, the field trashes such as tissue are removed, reclaim the supernatant containing target peptide.

Embodiment

< embodiment 1: utilize Ser1asRNA to suppress the expression > of Ser1 gene

(purpose)

Prove to utilize the gene expression that can suppress the sericin 1 (Ser1) as middle part silk glands specific protein of the asRNA for Ser1 gene.

(method)

1. the making of silk glands specific expression vector in the middle part of silkworm

Expression vector that can be specific expressed as silk glands in the middle part of silkworm, makes the Ser1asRNA expression vector expressing the asRNA (being designated as " Ser1asRNA ") for Ser1 gene and expresses the shRNA for Ser1 gene and (be designated as " Ser1shRNA ". Should illustrating, the shRNA of this specification also includes the longdsRNA with hairpin structure) Ser1shRNA expression vector.

(1) making of UAS skeleton plasmid

Using pAmCyan1-N1 (Takara) as template, AmCyankozakU (sequence numbering 23) and SV40PCRL (sequence numbering 24) is carried out pcr amplification as primer pair, inserts pZErO-2 carrier (lifetechnologies). It can be used as AmCyan/pZero2.

It addition, using pEYFP-N1 (clonetech) as template, EYFPkozakU (sequence numbering 25) and SV40PCRL is used as primer pair and carries out pcr amplification, insert pZErO-2 carrier (lifetechnologies). It can be used as EYFP/pZero2.

NcoI-NotI is utilized to be cut off by AmCyan/pZero2 and EYFP/pZero2, pBac [SerUAS/3xP3EGFP] (TatematsuK.etal. is inserted by the NcoI-NotI fragment that will obtain, 2010, TransgenicResearch, 19 (3): 473-87) NcoI-NotI position, is thus replaced with AmCyan or EYFP by marker gene by EGFP. Using these plasmids as pBac [SerUAS/3xP3AmCyan] or pBac [SerUAS/3xP3EYFP].

(2) clone of Ser1 fragment

From silkworm, the middle part silk glands in the 6th day 5 ages of in vain/C system extracts total serum IgE (IsogenNipponGene). Use oligodTprimer (Promega) and reverse transcriptase (ReverTraAceTOYOBO) that total serum IgE is carried out reverse transcription, obtain cDNA. Using the cDNA that obtains as template, use SpeSer1B130U23 (sequence numbering 26) and BlnSer1B1636L23 (sequence numbering 27) and BlnSer1B130U23 (sequence numbering 28) and these 2 kinds of primer pairs of SpeSer1B1636L23 (sequence numbering 29) to carry out PCR, obtain the 5 ' of the Ser1 fragment held. Using these nucleic acid fragments as 5 ' Ser1SB and 5 ' Ser1BS.

Additionally, same using the cDNA obtained as template, use SpeSer1B1531U24 (sequence numbering 30) and BlnSer1B3108L23 (sequence numbering 31) and BlnSer1B1531U24 (sequence numbering 32) and these 2 kinds of primer pairs of SpeSer1B3108L23 (sequence numbering 33) to carry out PCR, obtain the 3 ' of the Ser1 fragment held. Using these nucleic acid fragments as 3 ' Ser1SB and 3 ' Ser1BS.

(3) making of shRNA connector

Make the connector of the loop section being equivalent to shRNA. Using the genomic DNA of white for silkworm/C system as template, primer pair uses BlnA3intU (sequence numbering 34) and SpeA3IntL (sequence numbering 35) that the intron of actin 3 is carried out pcr amplification. Using the nucleic acid fragment that obtains as connector A3intBS.

(4) making of Ser1asRNA expression vector

The BlnI position of the pBac [SerUAS/3xP3AmCyan] made in above-mentioned (1) by BlnI become 5 ' hold in the way of insert 5 ' Ser1SB. Using this plasmid as pBac [SerUAS-5 ' asSer1/3xP3AmCyan] (Fig. 3 A). PBac [SerUAS-5 ' asSer1/3xP3AmCyan] is 5 ' the Ser1asRNA expression vectors of expression 5 ' Ser1asRNA, this 5 ' Ser1asRNA using 5 ' end regions of the Ser1 gene that do not contain repetitive sequence region as target site.

It addition, the BlnI position of pBac [SerUAS/3xP3EYFP] by BlnI become 5 ' hold in the way of insert 3 ' Ser1SB. Using this plasmid as pBac [SerUAS-3 ' asSer1/3xP3EYFP] (Fig. 3 B)). PBac [SerUAS-3 ' asSer1/3xP3EYFP] is 3 ' the Ser1asRNA expression vectors of expression 3 ' Ser1asRNA, and the repetitive sequence region that the 3 ' ends at Ser1 gene are existed by this 3 ' Ser1asRNA is as target site. 3 ' Ser1asRNA expression vectors, are equivalent to the 2nd subunit as the asRNA expression vector of effective ingredient of the expression of exogenous genes reinforcing agent containing the present invention.

(5) making of Ser1shRNA expression vector

The BlnI position of 5 ' Ser1asRNA expression vectors by BlnI position become 5 ' hold in the way of insert connector A3intBS, further by BlnI position become 5 ' hold in the way of insert 5 ' Ser1BS. Using this plasmid as pBac [SerUAS-5 ' dsSer1/3xP3AmCyan] (Fig. 4 A). PBac [SerUAS-5 ' dsSer1/3xP3AmCyan] is 5 ' the Ser1shRNA expression vectors of expression 5 ' Ser1shRNA, this 5 ' Ser1shRNA using 5 ' end regions of Ser1 gene as target site.

It addition, the BlnI position of 3 ' Ser1asRNA expression vectors by BlnI position become 5 ' hold in the way of insert connector A3intBS, further by BlnI position become 5 ' hold in the way of insert 3 ' Ser1BS. Using this plasmid as pBac [SerUAS-3 ' dsSer1/3xP3EYFP] (Fig. 4 B). PBac [SerUAS-3 ' dsSer1/3xP3EYFP] is 3 ' the Ser1shRNA expression vectors of expression 3 ' Ser1shRNA, and the repetitive sequence region that 3 ' ends of Ser1 gene are existed by this 3 ' Ser1shRNA is as target site.

2. the making of gene recombinaton silkworm

(1) importing of expression vector

The pBac as Ser1asRNA expression vector [SerUAS-5 ' asSer1/3xP3AmCyan] that will make in above-mentioned " the 1. making of silk glands specific expression vector in the middle part of silkworm " respectively, pBac [SerUAS-3 ' asSer1/3xP3EYFP] and as Ser1shRNA expression vector pBac [SerUAS-5 ' dsSer1/3xP3AmCyan] and pBac [SerUAS-3 ' dsSer1/3xP3EYFP] individually with the helper plasmid pHA3PIG (TamuraT.etal. expressing transposon transferring enzyme, 2000, NatureBiotechnology, 18, 81-84) mix with the ratio of 1:1, it is injected in the silkworm egg of w1-pnd system of after laying eggs 2～8 hours. ovum after injection, under humidification state, hatches until hatching at 25 DEG C. the larva of hatching, utilizes man-made feeds (SilkMate original seed 1-3 S in age, Japan's agricultural production work) to raise full age in the receptacle of 25～27 DEG C. exchange man-made feeds (UchinoK.etal., 2006, JInsectBiotechnolSericol, 75:89-97) in every 2～3 days. after emergence, carry out sib mating. presence or absence according to the fluorescence as the eye caused by the respective 3xP3EYFP label selecting label or 3xP3AmCyan label selects the F1 ovum obtained, and obtains gene recombinaton man mori system.

(2) with the copulation of middle part silk glands GAL4 system

By obtained to above-mentioned " importing of (1) expression vector " each gene recombinaton man mori system every kind 3 systematically with shown in Fig. 5, there is pBacSer-proGAL4/3xP3DsRed2 (TatematsuK.etal., 2010, TransgenicResearch, 19 (3): 473-87) gene recombinaton silkworm systematic mating. Herein, pBacSer-proGAL4/3xP3DsRed2 is the middle part silk glands GAL4 expression vector of the middle part specific expressed GAL4 gene of silk glands, is equivalent to the 1st subunit as the asRNA expression vector of effective ingredient of the expression of exogenous genes reinforcing agent containing the present invention. In F1 individuality, the presence or absence choice of the fluorescence of the eye caused by 3xP3EYFP label or 3xP3AmCyan label and 3xP3DsRed2 label has Ser1asRNA expression vector or Ser1shRNA expression vector (the 2nd subunit) and pBacSer-proGAL4/3xP3DsRed2 (the 1st subunit) both systems, obtains the gene recombinaton silkworm of middle part specific expressed Ser1asRNA or Ser1shRNA of silk glands. Owing to every kind 3 systematically uses the gene recombinaton silkworm with the 2nd subunit, therefore, the gene recombinaton silkworm of specific expressed Ser1asRNA or Ser1shRNA of middle part silk glands also respectively obtains every kind of 3 systems.

3.Ser1mRNA's is quantitative

Use the gene recombinaton silkworm of specific expressed Ser1asRNA or Ser1shRNA of middle part silk glands made by above-mentioned " 2. the making of gene recombinaton silkworm ", the expression inhibiting effect of the Ser1 gene of the middle part silk glands of each gene recombinaton silkworm is verified.

Extract middle part silk glands from the larva in the 6th day 5 ages of each gene recombinaton silkworm, use ISOGEN (NipponGene) to extract total serum IgE. Use oligodT primer (Promega) and reverse transcriptase (ReverTraAceTOYOBO) that total serum IgE carries out reverse transcription, prepare cDNA. Using the cDNA that obtains as template, the primer pair of Ser1LC3364 (sequence numbering 36) and Ser1LC3528L (sequence numbering 37) is used to pass through LightCyclerFastStartDNAMasterSYBRGreen (Roche) quantitative PCR carrying out the mRNA of Ser1. Interior mark uses the primer pair of rp49LCF2 (sequence numbering 38) and rp49LCR1 (sequence numbering 39), carries out quantitative PCR equally.

The checking of 4.Ser1 albumen quality

The gene recombinaton silkworm using specific expressed Ser1asRNA or Ser1shRNA of middle part silk glands made by above-mentioned " 2. the making of gene recombinaton silkworm " confirms the synthesis also suppressing Ser1 protein at protein level. Raise each gene recombinaton silkworm, after obtaining cocoon, add 50mMTris-HCl (the PH8.0)/8M carbamide/10mMDTT of 1mL to 10mg cocoon, hatch 5 minutes at 80 DEG C, thus extract cocoon protein. Cocoon protein 32.5 μ L is added the NuPAGESampleReducingAgent (Invitrogen) of the NuPAGELDSSampleBuffer (Invitrogen) and 5 μ L of 12.5 μ L, 70 DEG C of 10 minutes SDSization of heating. After using 4%SDS-PAGE gel that the sample of SDSization is carried out electrophoresis, use EZstainaqua (ATTO) dyeing.

(result)

The result of above-mentioned " 3.Ser1mRNA's is quantitative " is shown in Fig. 6. Relative value when the figure shows the Ser1mRNA amount of transcribing of the gene recombinaton silkworm (system that the 2nd subunit of Fig. 6 is represented by "-") of negative control as 1. The gene recombinaton silkworm with 5 ' Ser1shRNA expression vectors, the gene recombinaton silkworm with 3 ' Ser1shRNA expression vectors, there is the gene recombinaton silkworm of 5 ' Ser1asRNA expression vectors and there is arbitrary group of the gene recombinaton silkworm expression all confirming to suppress Ser1 gene of 3 ' Ser1asRNA expression vectors. The average relative value that the Ser1mRNA of 3 systems of each group (being equivalent to the 2nd subunit 1,2,3) measures is shown in top.

In the asRNA being made up of, being formed without intramolecular duplex RNA region single stranded RNA, express the group of 5 ' Ser1asRNA, as shown in conventional theory, compared with 2 groups that express the shRNA forming intramolecular duplex RNA region, the expression inhibiting effect of Ser1 gene is non-normally low. On the other hand, expressing the repetitive sequence region of Ser1 as the group of 3 ' Ser1asRNA of target site, although being asRNA, but there is the Ser1 gene expression inhibition effect of more than shRNA. This result shows the repetitive sequence region of the Ser1 protein asRNA as target site, with the gene expression that other regions effectively suppress at middle part silk glands Ser1 protein compared with the situation of target site.

The result of above-mentioned " checking of 4.Ser1 albumen quality " is shown in Fig. 7. The position of each cocoon protein is shown in the right side of SDS-PAGE. Ser1 protein passes through alternative splicing, and 2 bands different as molecular weight represent. To only have the gene recombinaton man mori system (UAS-of the 1st subunit; GAL4+) and not there is the 1st subunit and only there is the gene recombinaton man mori system (UAS3 of the 2nd subunit; GAL4-) as comparison.

Also the result same with the experiment of mRNA level in-site is obtained at protein level. That is, the whole gene recombinaton silkworm beyond comparison confirming, the synthesis of Ser1 protein is suppressed. On the other hand, as the Ser3 protein of identical cocoon protein, FibH chain protein synthetic quantity with compare as same degree. This shows that asRNA or the shRNA specificity expressed by Ser1asRNA expression vector or shRNA expression vector suppresses the expression of Ser1 gene.

< embodiment 2: utilize the asRNA for FibH chain gene to suppress the expression > of FibH gene

(purpose)

Embodiment 1 demonstrates the expression inhibiting effect of the asRNA of the gene for middle part silk glands specific protein Ser1. Therefore, this example demonstrates that, for also being able to suppress the expression of FibH gene also with the asRNA in the repetitive sequence region for FibH gene as fibroin albumen H (FibH) chain of rear portion silk glands specific protein.

(method)

1. the making of silkworm rear portion silk glands specific expression vector

Expression vector that can be specific expressed as silkworm rear portion silk glands, makes the Fib-HasRNA expression vector expressing the asRNA for FibH chain gene.

(1) making of UAS skeleton plasmid

BsmBIadapU (sequence numbering 40) and BsmBIadapL (sequence numbering 41) is annealed and makes BsmBI joint. BlnI position insertion BsmBI joint at the pBac [SerUAS/3xP3AmCyan] that embodiment 1 makes. Using the plasmid that obtains as pBac [SerUAS-BsmBI/3xP3AmCyan].

(2) clone in the repetitive sequence region of FibH chain

The genomic DNA of the large-scale manufacture system of silkworm is carried out PCR, the repetitive sequence region of amplification FibH chain gene by the primer pair utilizing FibHU (sequence numbering 42) and FibHL (sequence numbering 43). Using from 2 fragments of the multiple Piece Selection obtained as FibH1, FibH2. Utilize BsmBI to cut off FibH1 (1196bp) and FibH2 (1433bp), insert antisense strand at the BsmBI position of above-mentioned pBac [SerUAS-BsmBI/3xP3AmCyan] in the direction expressed. Using the plasmid that obtains as pBac [SerUAS-asFib1/3xP3AmCyan] (Fig. 8 A) and pBac [SerUAS-asFib2/3xP3AmCyan] (Fig. 8 B). These plasmids are all the Fib-HasRNA expression vectors of asRNA (being designated as " the Fib-HasRNA ") coding DNA containing the repetitive sequence region for FibH chain gene, are equivalent to the 1st subunit as the asRNA expression vector of effective ingredient of the expression of exogenous genes reinforcing agent containing the present invention.

2. the making of recombinant silkworm

(1) importing of expression vector

PBac [SerUAS-asFib1/3xP3AmCyan] and pBac [SerUAS-asFib2/3xP3AmCyan] is mixed with the ratio of 1:1 with above-mentioned helper plasmid pHA3PIG respectively, is injected to the silkworm egg of the w1-pnd system of after laying eggs 2～8 hours. Ovum after injection is hatched until hatching at 25 DEG C under humidification state. The larva of hatching utilizes man-made feeds (SilkMate original seed 1-3 S in age, Japan's agricultural production work) to raise full age in the receptacle of 25～27 DEG C. Man-made feeds exchange in every 2～3 days. After emergence, carry out sib mating. The presence or absence of the fluorescence of the eye caused by 3xP3AmCyan selects the F1 ovum obtained, and obtains gene recombinaton man mori system. For the gene recombinaton silkworm of 2 groups of FibH1 and FibH2, select 2 systems and 3 systems respectively, for the copulation with ensuing rear portion silk glands GAL4 system.

(2) with the copulation of rear portion silk glands GAL4 system

By gene recombinaton man mori system derived above with there is pBac [BmFibHL-GAL4/3xP3-DsRed] (SezutsuH.etal, 2009, JournalofInsectBiotechnologyandsericology, 78,1-10) gene recombinaton silkworm systematic mating. PBac [BmFibHL-GAL4/3xP3-DsRed] (Fig. 9) is the rear portion silk glands GAL4 expression vector being connected to GAL4 gene in the downstream in FibH promoter that rear portion silk glands is active, is equivalent to the 1st subunit as the asRNA expression vector of effective ingredient of the expression of exogenous genes reinforcing agent containing the present invention. In F1 individuality, the presence or absence choice of the fluorescence of the eye caused by 3xP3AmCyan label and 3xP3DsRed2 label has Fib-HasRNA expression vector (the 2nd subunit) and pBac [BmFibHL-GAL4/3xP3-DsRed] (the 1st subunit) both systems, obtains the gene recombinaton silkworm of the specific expressed asRNA for FibH chain of rear portion silk glands.

3.FibH chain mRNA's is quantitative

Using the gene recombinaton silkworm of the specific expressed Fib-HasRNA of rear portion silk glands made by above-mentioned " 2. the making of gene recombinaton silkworm ", the inhibition expressed for the FibH chain gene of rear portion silk glands is verified.

Extract rear portion silk glands from the larva in the 6th day 5 ages of each gene recombinaton silkworm, use ISOGEN (NipponGene) to extract total serum IgE. Use oligodT primer (Promega) and reverse transcriptase (ReverTraAceTOYOBO) that total serum IgE carries out reverse transcription, prepare cDNA. Using the cDNA that obtains as template, use the primer pair of FibHLCU (sequence numbering 44) and FibHLCL (sequence numbering 45), by LightCyclerFastStartDNAMasterSYBRGreen (Roche), carry out the quantitative PCR of the mRNA of FibH chain. Interior mark uses the primer pair of rp49LCF2 and rp49LCR1, carries out quantitative PCR equally.

The checking of 4.FibH catenin quality

Use the gene recombinaton silkworm of the specific expressed Fib-HasRNA of rear portion silk glands made by above-mentioned " 2. the making of gene recombinaton silkworm ", be also carried out confirming that the synthesis of FibH chain protein is suppressed at protein level. Extract silk glands from the larva in the 6th day 5 ages of each gene recombinaton silkworm, be separated into rear portion silk glands and middle part silk glands. Every to rear portion silk glands or middle part silk glands 1 is joined in 20mMTris-HCl (the pH8.0)/8M carbamide/2%SDS/25mMDTT of 5mL, overnight, vibrate under room temperature, extract silk glands protein. Although FibH chain gene is specific expressed at rear portion silk glands, but the reason being also carried out verifying for the protein of middle part silk glands is to be transferred to middle part silk glands after synthesizing in the silk glands of rear portion due to FibH chain protein, is therefore not only present in rear portion silk glands and exists in middle part silk glands. H is added to silk glands protein 5 μ L₂O27.5 μ L, 12.5 μ L the NuPAGESampleReducingAgent (Invitrogen) of NuPAGELDSSampleBuffer (Invitrogen) and 5 μ L, 10 minutes SDSization of heating at 70 DEG C. After using 4%SDS-PAGE gel to carry out electrophoresis in the sample of SDSization, EZstainaqua (ATTO) is utilized to dye.

(result)

The result of above-mentioned " 3.FibH chain mRNA's is quantitative " is shown in Figure 10. Relative value when the figure shows the FibH chain mRNA amount of transcribing of gene recombinaton silkworm (system that the 2nd subunit of Figure 10 is represented by "-") of comparison as 1. There is gene recombinaton silkworm FibH1 and the FibH2 of Fib-HasRNA expression vector, all confirm the expression inhibiting of FibH chain gene. This result shows that the asRNA in the repetitive sequence region for silkworm silk glands protein is not only effective to middle part silk glands specific protein, and is also effective to rear portion silk glands specific protein.

The result of above-mentioned " checking of 4.FibH catenin quality " is shown in Figure 11. The position of FibH chain protein and Ser1 protein is shown in the right side of SDS-PAGE. In vain/C system is the non-recombinant silkworm on the basis of the gene recombinaton silkworm becoming the present embodiment. Nd system is the abruptly-changing system expressing low silkworm of FibH chain gene, and additionally Nd-sD system is the abruptly-changing system expressing low silkworm of FibL chain gene. Should illustrating, Nd system and Nd-sD system, by the degeneration of rear portion silk glands, the expression of FibH chain and FibL chain gene reduces. Gene recombinaton man mori system (UAS-; GAL4+) it is only there is the 1st subunit (GAL4) and not there is the silkworm of the 2nd subunit (UAS-FibHasmRNA). These are the comparisons of the present embodiment.

Also the result same with the experiment of mRNA level in-site is obtained at protein level. That is, in there is the gene recombinaton silkworm of 2 subunits of Fib-HasRNA expression vector, the FibH catenin quality of the middle part silk glands of any system with compare (UAS-; GAL4+) compare and all substantially reduce. This shows, is suppressed by the synthesis expressing FibH chain protein of Fib-HasRNA at rear portion silk glands, it does not have synthesize new FibH chain protein, is finally reduced by the FibH catenin quality of rear portion silk glands inner chamber extrusion to middle part silk glands inner chamber.

On the other hand, as the Ser1 protein of identical silk glands protein synthetic quantity in the silk glands of middle part with compare (UAS-; GAL4+) it is same degree, also shows that the Fib-HasRNA specificity expressed by Fib-HasRNA expression vector suppresses the expression of Fib-H chain gene at protein level.

< embodiment 3: utilize Ser1asRNA and Fib-HasRNA to suppress the gene expression > of Ser1 and FibH chain

(purpose)

Prove to combine the asRNA in the repetitive sequence region for each gene of Ser1 and FibH, when expressing, also be able to suppress the expression of Ser1 gene and FibH chain gene simultaneously simultaneously.

(method)

1. the making of silk glands and rear portion silk glands expression vector in the middle part of silkworm

(1) Fib-HasRNA and Ser1asRNA expresses the making of UAS carrier (UAS-3 ' Ser1-FibH1asRNA expression vector) simultaneously

The BlnI position of 3 ' Ser1SB insertion pBac [SerUAS-asFibH1/3xP3AmCyan] prepared by embodiment 1, and making pBac [SerUAS-3 ' asSer1_asFibH1/3xP3AmCyan] (Figure 12 A). This plasmid is the expression vector (UAS-3 ' Ser1-FibH1asRNA expression vector) configuring 3 ' Ser1asRNA coding DNAs and Fib-HasRNA coding DNA in the downstream tandem of UAS promoter, is equivalent to the 2nd subunit as the asRNA expression vector of effective ingredient of the expression of exogenous genes reinforcing agent containing the present invention.

(2) making of & rear portion, middle part silk glands GAL4 expression vector

AscI.adU (sequence numbering 46) and AscI.adL (sequence numbering 47) is annealed, makes AscI joint. AscI joint is inserted the AscI position of Fig. 9 pBac [BmFibHL-GAL4/3xP3-DsRed] represented, makes pBacFibH-proGAL4_AscI-BlnI/3xP3DsRed2. Insert the AscI-SpeI fragment of pBacSer-proGAL4/3xP3DsRed2 further at the AscI-BlnI position of this plasmid, be produced on & rear portion, middle part silk glands GAL4 expression vector and the pBacFibH-proGAL4_Ser-proGAL4/3xP3DsRed2 (Figure 12 B) of middle part and rear portion silk glands expression GAL4 gene. This expression vector is equivalent to the 1st subunit as the asRNA expression vector of effective ingredient of the expression of exogenous genes reinforcing agent containing the present invention.

2. the making of recombinant silkworm

(1) importing of expression vector

The helper plasmid pHA3PIG that pBacFibH-proGAL4_Ser-proGAL4/3xP3DsRed2 as & rear portion, middle part silk glands GAL4 expression vector and the pBac as UAS-3 ' Ser1-FibH1asRNA expression vector [SerUAS-3 ' asSer1_asFibH1/3xP3AmCyan] individually records with embodiment 1 respectively is mixed with the ratio of 1:1, is injected to the silkworm egg of the w1-pnd system of after laying eggs 2～8 hours respectively. Ovum after injection is hatched until hatching at 25 DEG C under humidification state. The larva of hatching utilizes man-made feeds (SilkMate original seed 1-3 S in age, Japan's agricultural production work) to raise full age in the receptacle of 25～27 DEG C. Man-made feeds exchange in every 2～3 days. After emergence, carry out sib mating. From the F1 obtained, the presence or absence of the fluorescence of the eye caused by 3xP3DsRed2 label selects injection to have & rear portion, the middle part silk glands GAL4 system of & rear portion, middle part silk glands GAL4 expression vector, additionally, the presence or absence of the fluorescence of the eye caused by 3xP3AmCyan label selects injection to have UAS-3 ' the Ser1-FibH1asRNA system of UAS-3 ' Ser1-FibH1asRNA expression vector, obtains gene recombinaton man mori system.

(2) copulation of middle part & rear portion silk glands GAL4 system and UAS-3 ' Ser1-FibH1asRNA system

By & rear portion, middle part silk glands GAL4 system and UAS-3 ' Ser1-FibH1asRNA systematic mating. Additionally, as comparison use, what embodiment 1 used has a middle part gene recombinaton man mori system of silk glands GAL4 expression vector pBacSer-proGAL4/3xP3DsRed2 or the gene recombinaton man mori system with rear portion silk glands GAL4 expression vector pBac [BmFibHL-GAL4/3xP3-DsRed] that embodiment 2 uses and Ser1-FibH1asRNA systematic mating. In F1 individuality, the presence or absence choice of the fluorescence of the eye caused by 3xP3DsRed2 label and 3xP3AmCyan label has the system of middle part & rear portion silk glands GAL4 system and UAS-3 ' Ser1-FibH1asRNA system, and in the middle part of obtaining, silk glands and/or rear portion silk glands express the gene recombinaton silkworm of Fib-HasRNA and Ser1asRNA simultaneously.

The checking of 3.Ser1 albumen quality and FibH catenin quality

Use the gene recombinaton silkworm made by above-mentioned " 2. the making of gene recombinaton silkworm " that Ser1 albumen quality and FibH catenin quality are verified. Extract silk glands from the larva in the 6th day 5 ages of each gene recombinaton silkworm, be separated into rear portion silk glands and middle part silk glands. Add 20mMTris-HCl (the pH8.0)/8M carbamide/2%SDS/25mMDTT to 5mL by every to rear portion silk glands or middle part silk glands 1, overnight, vibrate under room temperature, extract silk glands protein. H is added to silk glands protein 5 μ L₂O27.5 μ L, 12.5 μ L the NuPAGESampleReducingAgent (Invitrogen) of NuPAGELDSSampleBuffer (Invitrogen) and 5 μ L, 70 DEG C of 10 minutes SDSization of heating. After using 4%SDS-PAGE gel that the sample of SDSization is carried out electrophoresis, CBC is utilized to dye.

(result)

Figure 13 illustrates result. When 1st subunit uses rear portion silk glands to express with GAL4 carrier, and compare (UAS-; GAL4+) the FibH catenin Mass lost of middle part silk glands is compared. This is consistent with the result of embodiment 2, it addition, when the 1st subunit uses middle part silk glands to express with GAL4 carrier, the Ser1 protein of middle part silk glands disappears substantially. This shows by middle part silk glands expression GAL4 carrier, expresses at middle part silk glands Ser1asRNA, it is suppressed that the expression of Ser1 gene. On the other hand, when the 1st subunit uses & rear portion, middle part silk glands to express with GAL4 carrier, the both sides at middle part and rear portion silk glands express Fib-HasRNA and Ser1asRNA. Thus, Ser1 gene and FibH chain gene are suppressed simultaneously and efficiently and express.

< embodiment 4: utilize Ser1asRNA to strengthen the expression > of allogenic gene

(purpose)

According to embodiment 1 and 3, it was demonstrated that inhibited the expression of Ser1 gene by Ser1asRNA. Therefore, it was demonstrated that by the expression inhibiting of Ser1 gene, it is possible to strengthen the expression of allogenic gene.

(method)

1. the making of expression of exogenous genes carrier

Utilize NcoI-NotI UAS skeleton plasmid AmCyan/pZero2 embodiment 1 made to cut off, cut out AmCyan genetic fragment. The fragment obtained is inserted pBac [SerUAS-ser_int-EGFP/3xP3EGFP] (Figure 14 A) (TatematsuK.etal., 2010, TransgenicResearch, 19 (3): 473-87) NcoI-NotI position. Thus the marker gene of pBac [SerUAS-ser_int-EGFP/3xP3EGFP] is replaced with AmCyan by EGFP. Using the UAS-EGFP expression vector that obtains as pBacSerUAS-ser_intEGFP/3xP3AmCyan (Figure 14 B).

2. the making of recombinant silkworm

(1) importing of expression of exogenous genes carrier

The helper plasmid pHA3PIG that UAS-EGFP expression vector pBacSerUAS-ser_intEGFP/3xP3AmCyan made by above-mentioned " 1. the making of expression of exogenous genes carrier " records with embodiment 1 is mixed with the ratio of 1:1, is injected to the silkworm egg of the w1-pnd system of after laying eggs 2～8 hours. Ovum after injection is hatched until hatching at 25 DEG C under humidification state. The larva of hatching utilizes man-made feeds (SilkMate original seed 1-3 S in age, Japan's agricultural production work) to raise full age in the receptacle of 25～27 DEG C. Man-made feeds exchange in every 2～3 days. After emergence, carry out sib mating. The presence or absence of the fluorescence of the eye caused by 3xP3AmCyan label selects the F1 obtained, and obtains gene recombinaton man mori system. Using this gene recombinaton man mori system as UAS-EGFP system.

(2) copulation of middle part silk glands GAL4 system, 3 ' Ser1asRNA systems and UAS-EGFP system

To there is the pBacSer-proGAL4/3xP3DsRed2 middle part silk glands GAL4 system as middle part silk glands expression GAL4 carrier, (being equivalent to the 1st subunit system) and UAS-3 ' the SerasRNA system with 3 ' Ser1asRNA expression vector pBac [SerUAS-3 ' asSer1/3xP3EYFP], (being equivalent to the 2nd subunit system) and ", (1) importing of expression of exogenous genes carrier " the UAS-EGFP system with pBacSerUAS-ser_intEGFP/3xP3AmCyan that obtains carries out copulation. as comparing use, above-mentioned middle part silk glands GAL4 system is carried out copulation with UAS-3 ' the SershRNA system and above-mentioned UAS-EGFP system with 3 ' Ser1shRNA expression vector pBac [SerUAS-3 ' dsSer1/3xP3EYFP]. it addition, as negative control use, only above-mentioned middle part silk glands GAL4 system and above-mentioned UAS-EGFP system are carried out copulation. label based on respective expression vector selects single individuality have the system of 3 kinds of expression vectors or have the negative control system of 2 kinds of expression vectors according to the presence or absence of the fluorescence of eye. thus, obtain the system of the expression inhibiting of the middle part specific expressed EGFP gene of silk glands and generation Ser1 gene, and the system of the expression inhibiting of Ser1 gene does not occur.

3. the EGFP protein of silk glands is quantitative

Use each gene recombinaton silkworm made by above-mentioned " 2. the making of gene recombinaton silkworm " that EGFP albumen quality is verified. Extract middle part silk glands, every 1 PBS (pH7.2)/1%Tween20/0.05% Hydrazoic acid,sodium salt adding 10mL to from the larva in the 6th day 5 ages, extract water soluble protein by vibrating under room temperature 24 hours. The water soluble protein extracting solution obtained is centrifuged 10 minutes with 2000 × g, reclaims supernatant. Reacti-BindAnti-GFPCoatedPlates (PIERCE) is utilized to measure the EGFP protein concentration in the water soluble protein that supernatant contains. Specifically, Reacti-BindAnti-GFPCoatedPlates adds 100 μ L of supernatant liquid, left at room temperature 1 hour. After utilizing PBS/0.05%Tween20 to clean 3 times, add horseradishperoxidase-conjugatedanti-GFPantibody (RocklandImmunochemicals), left at room temperature 1 hour. After utilizing PBS/0.05%Tween20 to clean 3 times, use TMBPeroxidaseEIASubstrateKit (Bio-Rad) to carry out chromogenic reaction, add 1N sulphuric acid and make reaction stop. Colour developing is utilized platereader (SpectraMax250; MolecularDevices) quantitative. Use restructuring GFP protein (TAKARABIO; Z2373N) serial dilutions (1-400pg/ μ L) makes standard curve.

(result)

Figure 15 illustrates the EGFP albumen quality of each individuality of each system. Express the system of 3 ' Ser1asRNA compared with comparison ("-" of Figure 15), there is the rising of the EGFP albumen quality of about 2 times. This shows that the expression of 3 ' Ser1asRNA, the Ser1 genes by expressing the repetitive sequence region for Ser1 gene at middle part silk glands is suppressed, and the expression of final allogenic gene (here for EGFP gene) strengthens. On the other hand, although utilizing 3 ' Ser1shRNA to suppress the expression of Ser1 gene also to increase EGFP albumen quality, but compared with 3 ' Ser1asRNA, its increase amount is low.

< embodiment 5: utilize Ser1asRNA and Fib-HasRNA's to express the expression > strengthening allogenic gene

(purpose)

It is verified and Ser1asRNA and Fib-HasRNA combination is expressed, suppress the expression of Ser1 gene and FibH gene such that it is able to more strengthen the expression of allogenic gene simultaneously.

(method)

1. the making of recombinant silkworm

The UAS-EGFP system with middle part & rear portion, the middle part silk glands GAL4 system of & rear portion silk glands GAL4 expression vector pBacFibH-proGAL4_Ser-proGAL4/3xP3DsRed2, UAS-3 ' Ser1-FibH1asRNA system and embodiment 4 preparation embodiment 3 prepared carries out copulation. It addition, as comparison use, only & rear portion, middle part silk glands GAL4 system and UAS-EGFP system are carried out copulation. The presence or absence of the fluorescence of the eye caused by label according to each expression vector is selected, and obtains the specific expressed EGFP of silk glands and middle part silk glands and/or rear portion silk glands expresses each 3 systems of gene recombinaton silkworm of Fib-HasRNA and Ser1asRNA simultaneously.

2. the EGFP protein of silk glands is quantitative

Use each gene recombinaton silkworm made by above-mentioned " 1. the making of gene recombinaton silkworm ", EGFP albumen quality is verified. Extract silk glands, every 1 PBS (pH7.2)/1%Tween20/0.05% Hydrazoic acid,sodium salt adding 20mL to from the larva in the 6th day 5 ages, extract water soluble protein by vibrating under room temperature 24 hours. The water soluble protein extracting solution obtained is centrifuged 10 minutes with 2000 × g, reclaims supernatant. Reacti-BindAnti-GFPCoatedPlates (PIERCE) is used to measure the EGFP protein concentration in the water soluble protein that supernatant contains. Specifically, Reacti-BindAnti-GFPCoatedPlates adds 100 μ L of supernatant liquid, left at room temperature 1 hour. After utilizing PBS/0.05%Tween20 to clean 3 times, add horseradishperoxidase-conjugatedanti-GFPantibody (RocklandImmunochemicals), left at room temperature 1 hour. After utilizing PBS/0.05%Tween20 to clean 3 times, use TMBPeroxidaseEIASubstrateKit (Bio-Rad) to carry out chromogenic reaction, add 1N sulphuric acid and make reaction stop. Colour developing is utilized platereader (SpectraMax250; MolecularDevices) quantitative. Use restructuring GFP protein (TAKARABIO; Z2373N) serial dilutions (1-400pg/ μ L) makes standard curve.

(result)

Figure 16 illustrates each individual EGFP albumen quality of each system. In the system expressing 3 ' Ser1asRNA and Fib-HasRNA, compared with the negative control of the expression not suppressing these genes ("-" of Figure 17), find high EGFP albumen quality at silk glands. This is it is shown that suppressed by respective asRNA by the synthesis of Ser1 and the FibH chain as middle part silk glands and the main protein of rear portion silk glands, and the expression of allogenic gene (here for EGFP gene) strengthens.

< embodiment 6: utilize the expression of Ser1asRNA and Fib-HasRNA to reduce and be mingled with protein G reatT.GreaT.GT

(purpose)

Confirm to hinder the gel of the extraction of target peptide to be mingled with protein (being mainly made up of) sericin and fibroin albumen to reduce by expressing Ser1asRNA and Fib-HasRNA.

(method)

From each 3 the extraction middle parts of the larva in the 6th day 5 ages of the system identical with the gene recombinaton silkworm that embodiment 5 makes and rear portion silk glands. Collect the middle part of 1 share and rear portion silk glands and be added into 4mLPBS, overnight overturning stirring at 4 DEG C. 4 DEG C, be centrifuged with 5000rpm, the supernatant that obtain is loaded 15mL pipe, 4 DEG C stand 48 hours. Make pipe turn upside down, measure the liquid measure fallen, calculate and fall relative to the liquid of original liquid measure a few percent.

(result)

Figure 17 illustrates result. Supernatant gelation in not suppressing the negative control of expression of Ser1 and FibH chain gene, white opacity. Finally, even if the extracting solution that turned upside down by pipe does not also fall completely. On the other hand, in the sample deriving from the system expressing Ser1asRNA and Fib-HasRNA, supernatant not gelation, keep liquid condition. When being turned upside down by pipe, the extracting solution of average 98% falls. These are it is shown that by utilizing Ser1asRNA and Fib-HasRNA to suppress Ser1 gene and the expression of FibH chain gene respectively, it is possible to effectively remove the gelation becoming silk glands extracting solution reason be mingled with protein.

Should illustrate, the whole publications quoted in this specification, patents and patent applications are quoted into this specification directly as reference.

Claims

1. the expression of exogenous genes reinforcing agent of a silkworm silk glands, it is characterized in that, containing making the antisense RNA expression vector of specific expressed at least one antisense RNA in sericin 1 antisense RNA, sericin 2 antisense RNA and sericin 3 antisense RNA of silk glands in the middle part of silkworm and/or the antisense RNA expression vector of silkworm rear portion silk glands specific expressed fibroin albumen H chain antisense RNA can be made as effective ingredient, wherein

Described sericin 1 antisense RNA contains and the sequence of base sequence complementary of at least one repetitive in repetitive sequence region of 628～1054 in the aminoacid sequence of the domestic silkworm silk glue protein 1 shown in coded sequence numbering 1,

Described sericin 2 antisense RNA contains and the sequence of base sequence complementary of at least one repetitive in repetitive sequence region of 41～117 or 685～1359 in the aminoacid sequence of the domestic silkworm silk glue protein 2 shown in coded sequence numbering 4,

Described sericin 3 antisense RNA contains and the sequence of base sequence complementary of at least one repetitive in repetitive sequence region of 141～978 and 1031～1178 in the aminoacid sequence of the domestic silkworm silk glue protein 3 shown in coded sequence numbering 8,

Described fibroin albumen H chain antisense RNA contains and the sequence of base sequence complementary of at least one repetitive in repetitive sequence region of 152～5203 in the aminoacid sequence of the silk fibroin protein H chain shown in coded sequence numbering 12.

2. expression of exogenous genes reinforcing agent according to claim 1, wherein, the repetitive of sericin 1 aminoacid sequence shown in sequence numbering 2 is constituted,

The repetitive of sericin 2 aminoacid sequence shown in sequence numbering 5 or 6 is constituted,

The repetitive of sericin 3 aminoacid sequence shown in sequence numbering 9 or 10 is constituted, additionally

The repetitive of fibroin albumen H chain aminoacid sequence shown in sequence numbering 13 is constituted.

3. expression of exogenous genes reinforcing agent according to claim 1, wherein,

Sericin 1 antisense RNA base sequence shown in sequence numbering 3 is constituted,

Sericin 2 antisense RNA base sequence shown in sequence numbering 7 is constituted,

Sericin 3 antisense RNA base sequence shown in sequence numbering 11 is constituted, additionally

Fibroin albumen H chain antisense RNA base sequence shown in sequence numbering 14 is constituted.

4. the expression of exogenous genes reinforcing agent according to any one of claims 1 to 3, wherein, described antisense RNA expression vector is made up of 2 subunits.

5. a gene recombinaton silkworm, it is characterized in that, there is the antisense RNA expression vector that can make specific expressed at least one antisense RNA in sericin 1 antisense RNA, sericin 2 antisense RNA and sericin 3 antisense RNA of silk glands in the middle part of silkworm and/or the fibroin albumen H chain antisense RNA expression vector that can make the silkworm rear portion specific expressed antisense RNA of silk glands, wherein

Described sericin 3 antisense RNA contains and the sequence of base sequence complementary of at least one repetitive in repetitive sequence region of 141～978 or 1031～1178 in the aminoacid sequence of the domestic silkworm silk glue protein 3 shown in coded sequence numbering 8,

Silk glands specific expressed antisense RNA in described silkworm rear portion contains and the sequence of base sequence complementary of at least one repetitive in repetitive sequence region of 152～5203 in the aminoacid sequence of the silk fibroin protein H chain shown in coded sequence numbering 12.

6. gene recombinaton silkworm according to claim 5, wherein,

The repetitive of sericin 1 aminoacid sequence shown in sequence numbering 2 is constituted,

The repetitive of sericin 3 aminoacid sequence shown in sequence numbering 9 or 10 is constituted, or

7. gene recombinaton silkworm according to claim 5, wherein,

Sericin 2 antisense RNA base sequence shown in sequence numbering 6 is constituted,

Sericin 3 antisense RNA base sequence shown in sequence numbering 9 is constituted, or

8. the gene recombinaton silkworm according to any one of claim 5～7, wherein, described antisense RNA expression vector is made up of 2 subunits.

9. gene recombinaton silkworm according to claim 8, wherein, described 2 subunits are present on different chromosomes.

10. the gene recombinaton silkworm according to any one of claim 5～9, has the expression of exogenous genes carrier that can express target allogenic gene.

11. the manufacture method of a gene recombinaton silkworm, it is characterised in that include the operation that silkworm is given the expression of exogenous genes reinforcing agent according to any one of Claims 1 to 4.

12. the method manufacturing the target peptide of allogenic gene coding in the silk glands of the gene recombinaton silkworm described in claim 10.