114 results about "Silk gland" patented technology

The invention relates to enhancer Hr3 adopting the nucleotide sequence shown in the SEQIDNO: 1. The enhancer Hr3 can be used for preparing and recombining heterologous protein by bombyx mori. The invention further relates to a recombination carrier containing the enhancer Hr3, and the recombination carrier is prepared in such a manner that the enhancer Hr3 is connected with a Nco1 restriction site at the upstream of a promoter of a carrier containing no enhancer through the Nco1 restriction site of the enhancer Hr3. In the invention, functional elements such as the first grade enhancer, activating transcription factor IE1, untranslated region sequences (5' UTR and 3' UTR) and the like which are expressed by intensifier are identified in the cellular level, and an efficient and stable sericin I expression system is built by integrating optimum elements on the basis, so that efficient expression recombination heterologous protein can be made of middle silk gland of transgenic bombyx mori.

The invention relates to a silkworm fibroin heavy-chain gene mutation method, which specifically comprises the step of: acting mRNA (Messenger Ribonucleic Acid) of a coded zinc-finger nuclease sequence on loci 1325-1362 of a silkworm fibroin heavy-chain gene shown as SEQ ID NO:1 to form target positions for recognizing zinc-finger nuclease, thereby obtaining a series of silkworm fibroin heavy-chain mutated genes; and the mutated sequence can be applied to preparation of sericin and extrinsic proteins. Mutants provided by the invention have the following advantages that: (1) a posterior division of silkgland of a fibroin heavy-chain gene mutant provided by the invention serious degrades, a cocoon shell only contains the sericin synthesized and secreted by a middle division of silkgland, if the mutated strains are utilized to transgenically express the extrinsic proteins, the expressed amount and the purity of the extrinsic proteins can be greatly increased, and thus, a brand-new useful genetic material is provided for the development of a silkworm fibroin bioreactor; and (2) the cocoon shell of the mutated strain provided by the invention only contains the sericin, and thus, a new source is provided for the large-scale production of the sericin.

The invention discloses a method for building a domestic silkworm silk gland biologic plant. A recombinant transgene vector which is provided with domestic silkworm silk protein specificity promoter controlled extraneous gene expression cassettes, an enhancer activity component, antibiotic resistant genes and fluorescent protein reporter genes and based on a piggyBAC transposon is established through gene engineering operations. The transgene vector is introduced into primarily laid eggs, special antibiotics are fed to hatched newly exuviated silkworms continuously until the fourth instar. In this way, silkworms which can resist the specific antibiotics and are provided with fluorescent protein reporter genes are obtained; target genes are detected; and transgene silkworms are manufactured by breeding. By adopting the silk gland tissues of the transgene silkworms, protein drugs can be obtained. With the method disclosed by the invention, transgene domestic silkworm silk gland plants can prepare protein drugs at low manufacture cost without potentially hazardous rhabdovirus. The method is safe and is practically valuable.

The invention relates to a nano-zinc oxide feeding silkworm rearing method for manufacturing high-performance silk and a product thereof. The nano-zinc oxide feeding silkworm rearing method includes steps of adding nano-zinc oxide into artificial feed of silkworms, feeding nano-zinc oxide into silk gland of silkworms by an absorbing and converting function of the silkworms and cocooning to obtain high-performance silk containing nano-zinc oxide. On the premise of not damaging natural quality of the silk, the silk is modified and has better mechanical performance and better sterilization and ultraviolet resistance property.

The invention discloses a method for synthesizing and secreting black widow spider dragline silk protein 1 through a bombyx mori silk gland bioreactor. The method includes the steps that firstly, pBac[3xP3-DsRed]-MaSp1 plasmids serving as a carrier for bombyx mori to synthesize and secrete black widow spider dragline silk protein 1 are established; then, plasmids and assistant plasmids are introduced into bombyx mori zygotes through microinjection, red fluorescence protein genes and black widow spider dragline silk protein 1 genes are introduced into bombyx mori genomes through the transposition characteristic of piggyBac transposons, stable inheritance and expression are performed, transgenic bombyx mori is obtained, mori moth selfing is performed to achieve homozygosis of black widow spider dragline silk protein 1 genes, and transgenic bombyx mori secreting black widow spider dragline silk protein 1 is bred. According to the method, transgenic bombyx mori is screened through fluorescence marker genes, and black widow spider dragline silk protein 1 is specifically synthesized and secreted through bombyx mori silk gland cells; the method is used for development and utilization of spider silk and can also be used for improving the mechanical performance of silk.

The invention relates to a transgenic method for improving silk yield. Genes promoting cell growth are specifically expressed in posterior silk glands of silkworms, silkworms with enlarged posterior silk glands are obtained, and the yield of silk is high. The invention provides genes promoting the growth of posterior silk glands and an expression system suitable for expressing the genes promotingthe growth of posterior silk glands at high specificity in the posterior silk glands of the silkworms. The invention provides novel means and a method for improving silk producing characters of the silkworm variety.

A modified human acidic fibroblast growth factor gene is disclosed. The nucleotide sequence of the gene is shown as the number 25 locus to the number 450 locus of SEQ ID NO.1. The sequence is designed according to codon preferences of bombyx mori, and can be normally expressed and translated in the bombyx mori. A recombinant vector containing the modified human acidic fibroblast growth factor gene is also disclosed. The recombinant vector comprises an enhanser hr3 and a secreting type sericin 1 promoter Ser1, specifically expresses a modified human acidic fibroblast growth factor in a middle silk gland cell of the bombyx mori, and secretes a recombinant protein to a sericin layer of silk. The obtained silk has bioactivity, has a function of promoting cell proliferation when the silk is directly used, and is a good medical material.

The invention discloses a gene for modifying a human acidic fibroblast growth factor applied to silkworm silk gland expression, and an expression system and application of the gene. The gene for modifying the human acidic fibroblast growth factor is represented by the 7-450 site of SEQ ID NO.1; the amino acid coded by the gene is represented by SEQ ID NO.2; the gene for modifying the human acidic fibroblast growth factor, a gene promoter of secretory type sericin 1 and a gene terminator of the secretory type sericin 1 form an expression box, and the expression box is expressed by using an enhancer hr3 in an enhanced manner; simultaneously, the expression system is constructed by accessing a piggyBac transposable arm and a fluorescent selected and marked gene. The expression system is capable of expressing and recombining the human acidic fibroblast growth factor in silkworm silk gland at high efficiency; furthermore, the expression system has biological activity and can be used for producing and recombining the human acidic fibroblast growth factor in a large scale, therefore, the expression system has good market prospects.

The present inventors produced transgenic silkworms which comprise a promoter of a DNA encoding a protein specifically expressed in the silk gland and a DNA encoding a recombinant antibody whose expression is regulated directly or indirectly by the promoter, and which secrete the recombinant antibody into the silk gland. The recombinant antibodies produced from the silk gland of the transgenic silkworms were confirmed to be active.

Transgenic silkworms comprising GFP whose expression is regulated by the sericin gene promoter were produced. Observation of the silk glands of the last instar larvae of the silkworms showed fluorescence only in the middle silk glands. GFP was secreted from middle silkgland cells from around the spinning stage, indicating that GFP moved into the gland lumen. Finally, GFP was spun into cocoon filaments, and cocoons containing large amounts of GFP were produced. Thus, by using the promoter region of the sericin gene, recombinant proteins can be produced in the middle silk glands. Furthermore, the recombinant proteins produced in the middle silk glands were readily secreted into the lumen of the middle silk glands.

The present invention provides a genetic engineering material for insects that enables a target protein to be purified easily, without requiring the use of recombinant baculovirus, while simultaneously providing a process for producing exogenous protein using that genetic engineering material. A gene recombinant silkworm is obtained by inserting an exogenous protein gene such as a cytokine gene coupled to a promoter that functions in silk glands into a silkworm chromosome. An exogenous protein such as a cytokine is then extracted and purified from the silk glands or cocoon of that silkworm or its offspring. A large amount of exogenous protein can be produced within silk gland cells, outside silk gland cells or in silk thread or a cocoon by inserting an expression gene cassette, in which the DNA sequence of the 3′ terminal portion and the DNA sequence of the 5′ terminal portion of fibroin H chain gene are fused to the exogenous protein gene, into silk gland cells and so forth.

The invention discloses a BmCP274 promoter of bombyx mori cuticular protein. The promoter consists of nucleotide sequences shown in SEQ ID No.1, can drive foreign proteins to specifically express at a front silk gland of the bombyx mori and can be used as a specific promoter at the front silk gland of the silkworm. The invention further discloses a recombinant expression vector containing the promoter, which provides a powerful tool for a next-step study on regulating ion channel protein expression at the front silk gland of the silkworm to affect the behavior of the bombyx mori and improve the performance of the silk fiber.

The invention relates to the field of silkworm cultivation processing and in particular relates to a method for producing sericin protein. The method for producing the sericin protein comprises the following steps: picking out middle silk glands of silkworms of five years in age; adding water into the middle silk glands, and homogenizing to obtain homogenate; centrifuging the homogenate to obtain supernate dissolved with sericin protein; drying the supernate, thus obtaining the sericin protein. Compared with a method that silkworm cocoon shells are crushed and then the sericin protein is extracted in the prior art, the method for producing the sericin protein has the advantages that the middle silk glands (massive sericin protein is contained in the middle silk glands) are directly acquired from the silkworms of five years in age while the silkworms do not need complete a cocooning process, and the middle silk glands are homogenized, centrifuged and dried to obtain the sericin protein, so that the production period of sericin protein is shortened.

As novel means for causing any recombinant protein produced by the silkworm middle silk gland to be secreted in cocoons and easily extracting the recombinant protein from the cocoons without modification of the conformation of the protein, there is provided a polynucleotide that in an expression cassette for expression of a recombinant protein by the silkworm middle silk gland, is functionally linked to a recombinant protein structure gene, which polynucleotide is one composed of a polynucleotide constructing the promoter regions of sericin 1 or sericin 2 gene and, functionally linked thereto, a polynucleotide constructing the homologous regions of baculovirus.

The present invention provides a genetic engineering material for insects that enables a target protein to be purified easily, without requiring the use of recombinant baculovirus, while simultaneously providing a process for producing exogenous protein using that genetic engineering material. A gene recombinant silkworm is obtained by inserting an exogenous protein gene such as a cytokine gene coupled to a promoter that functions in silk glands into a silkworm chromosome. An exogenous protein such as a cytokine is then extracted and purified from the silk glands or cocoon of that silkworm or its offspring. A large amount of exogenous protein can be produced within silk gland cells, outside silk gland cells or in silk thread or a cocoon by inserting an expression gene cassette, in which the DNA sequence of the 3′ terminal portion and the DNA sequence of the 5′ terminal portion of fibroin H chain gene are fused to the exogenous protein gene, into silk gland cells and so forth.

Transgenic silkworms comprising GFP whose expression is regulated by the sericin gene promoter were produced. Observation of the silk glands of the last instar larvae of the silkworms showed fluorescence only in the middle silk glands. GFP was secreted from middle silkgland cells from around the spinning stage, indicating that GFP moved into the gland lumen. Finally, GFP was spun into cocoon filaments, and cocoons containing large amounts of GFP were produced. Thus, by using the promoter region of the sericin gene, recombinant proteins can be produced in the middle silk glands. Furthermore, the recombinant proteins produced in the middle silk glands were readily secreted into the lumen of the middle silk glands.

The invention discloses a reconstructed human serum albumin gene suitable for cultivated silk gland expression and an expression system and application thereof. The reconstructed human serum albumin gene is as shown as the 7-1767th in SEQ ID NO.1, and the coded amino acid is as shown in SEQ ID NO.2. The reconstructed human serum albumin gene, a secretion type sericin 1 gene promoter and a secretion type sericin 1 gene terminator form an expression frame, an enhancer Hr3 is used for enhancing expression, and simultaneously a piggyBac transposon and a fluorescent screening marker gene construct an expression system. The expression system can highly effectively express recombined human serum albumin in cultivated silk gland, has biological activity, can be used for large scale production of recombined human serum albumin, and has good market prospect.

The invention discloses a bombyx mori fibroin heavy chain expression system for fibroin and sericin expressing a target protein, and a preparation method and application of the bombyx mori fibroin heavy chain expression system. The expression system contains a fibroin heavy chain promoter 3 at the 5' end and an expression cassette of a heavy chain gene poly(A) sequence at the 3' end. The system isused to express a foreign protein, through the identification and detection analysis of transgenic bombyx mori, it is found that the presence of EGFP is detected in both the silk gland and the silk,and EGFP is also distributed in the sericin layer in the silk, so that the result indicates that in the absence of the C-terminal, the N-terminal is not an essential factor for the formation of the silk, and the absence of the C-terminal can make the protein be distributed in the sericin layer. The analysis of the N-terminal of fibroin provides an experimental and theoretical basis for the silk formation mechanism of the silk, and is conducive to creating a really practical bombyx mori silk gland bioreactor, maintaining the sustainable development of the silk industry, and promoting the long-term development of sericulture and insect science in China.

The invention discloses a method for preparing a biological antibacterial organic fertilizer by using an immune induced biological technology. In the method, Super-strong immune protein synthesizing capability of silkworm is utilized. The method comprises the following steps of: synthesizing a large amount of sericin antimicrobial peptides through silk glands in the middle of nature silkworm with five years old, and synthesizing silk fibroin antimicrobial peptides through silk glands at the rear; and regulating and controlling silk fibroin genes by using a biological high active agent, and synthesizing and amplifying by using a solid-phase or liquid-phase biological technology to obtain the biological antibacterial organic fertilizer. The method is simple and easily learnt in biological synthetic route technology, low in production cost and high in yield controllability; the disease preventing capability reaches above 80 to 95 percent; the fertilizer is easily converted and is convenient for industrialized production; and the biological antibacterial organic fertilizer prepared by adopting the method is mixed with soil in agricultural use, roots are soaked into the fertilizer solution before planting, and the fertilizer has a strong antibacterial effect and efficient and broad-spectrum antibacterial advantages on infection caused by withering fusarium and pathogens such as Gram-positive bacteria, Gram-negative bacteria or bacillus subtilis, bacterial wilt and the like.

The invention discloses a domestic silkworm cuticle protein BmCP231 promoter which is formed by No.1 nucleotide to No.1 1457 nucleotide in the SEQ ID No. 1 or No.1 nucleotide to No.1 1511 nucleotide in the SEQ ID No. 1. The domestic silkworm cuticle protein BmCP231 promoter can drive the specific expression of the exogenous protein on the front silk gland of the domestic silkworm and can be used as the specific promoter of the front silk gland of the domestic silkworm. The invention further discloses a recombinant expression vector containing the domestic silkworm cuticle protein BmCP231 promoter. A powerful tool is provided for the next-step research on regulating the expression of the protein of the ion channel for the front silk gland of the domestic silkworm to affect the spinning behavior of the domestic silkworm and improve the silk fiber performance.

The invention discloses a process for preparing board-spectrum antibiotics by immune induction biotechnology, and particularly relates to a process for preparing board-spectrum antibiotics by immune induction biotechnical synthesis, which employs super immunoprotein synthesis ability of silkworms and includes: allowing fifth-instar silkworms to synthetize high-quantity sericin antibacterial peptides from a middle division of silkgland and silk fibroin antibacterial peptides from a posterior division of the silkgland; regulating silk protein genes with highly bioactive agent; and performing synthesis and amplification to obtain board-spectrum antibiotics by solid-phase and liquid-phase biotechnology. By the process, biotechnical synthetic routes are simple and easy to learn, production cost is low, productivity is highly controllable, disease resistance reaches more than 80%-95%, and achievement is easy to transform and convenient for industrial production. The antibacterial peptides prepared by the process can be mixed with oil and used for soaking roots, and have a higher bacterial effect and efficient and broad-spectrum antibacterial advantages to infections caused by wilt fusarium, gram-positive bacterium, gram-negative bacterium or pathogens such as Bacillus subtilis, bacterial wilt and the like.

The invention discloses a silkworm flat silk spinning device. The silkworm flat silk spinning device comprises a spinning flat, a spinning flat supporting column, a flat bracket and a flat bracket supporting column; the upper ends of the spinning flat and spinning flat supporting column are hinged through a balance fulcrum, the spinning flat supporting column at one side of the spinning flat is afirst supporting column, the lower end of the first supporting column is fixed on the flat bracket, the spinning flat supporting column at the other side of the spinning flat is a second supporting column, and the lower end of the second supporting column is directly in contact with the flat bracket; the area of the spinning flat is less than the area of the flat bracket, and the spinning flat islocated in the middle of the position directly above the flat bracket. The silkworm flat silk spinning device can be folded to be used and can also be detached, thereby effectively preventing silkwormfrom dropping onto the ground and causing the break of a silk gland, preventing the silkworm body from being contaminated, and saving the time and labor.

A transformed silkworm which has a polynucleotide encoding human collagen in its genomic DNA and produces recombinant human collagen as a part of proteins in its cocoon or silk gland; a process for producing human collagen by using this transformed silkworm; and a recombinant vector to be used in the constitution of the transformed silkworm. Since human collagen is collected from the cocoon discharged by this transformed silkworm or its silk gland, highly pure human collagen can be conveniently obtained in a large amount. Moreover, the recombinant human collagen produced by the transformed silkworm is a highly safe collagen which is free from any fear of the contamination with pathogens such as viruses or prions and shows no antigenicity on humans. Thus, it is usable in various industrial fields including medicines, foods and cosmetics.

The invention relates to an HLH transcriptional regulator BmHLH2 applied to special expression of domestic silkworm silk gland. The HLH transcriptional regulator BmHLH2 is obtained by a series of biotechnology clones such as analyses of an expression profile of silk gland genes, manufacturing, hybridization and data analysis of genetic chips and the like. The HLH transcriptional regulator obtained in the invention is a novel transcriptional regulator applied to the special expression of the domestic silkworm silk gland, fluorescent quantitative PCR experiment indicates that the transcriptional regulator has important links with the development of the silk gland and differentiation of silk gland cells; an intervention study on the BmHLH2 gene indicates that the HLH transcriptional regulator plays a very important role in regulating and controlling cell differentiation, specialization of organs and the special expression of genes. The research findings are of great significance in improving silk qualify and quantity of domestic silkworms and promoting the development of silk industry.

The invention relates to a modified human lactoferrin gene suitable for bombyx mori silk gland expression as well as an expression system adopting the modified human lactoferrin gene and applications.The modified human lactoferrin gene is as shown in SEQ ID NO.1, the amino acid coded by the gene is as shown in SEQ ID NO.2, the modified human lactoferrin gene, a secreting type sericin 1 gene promoter and a secreting type sericin 1 gene terminator form an expression cassette, an enhanser hr3 is adopted for enhancing the expression, meanwhile, a piggyBac transposition arm and a fluorescence selection marker gene are connected for constructing the expression system, and the expression system can efficiently express the recombinant human lactoferrin in bombyx mori silk gland, has biological activity, can be used for mass production of recombinant human lactoferrin, and has good market prospects.

The invention discloses a fluorescent silk making method based on spanish mackerel organ and folium mori composite carbon dots. The fluorescent silk making method comprises the following steps: (1) preparing biocompatibile composite fluorescent carbon dots, namely preparing and extracting fluorescent carbon dots from spanish mackerel organs and folium mori by using a hydrothermal synthesis method;(2) breeding silkworms, namely spraying the prepared water-soluble fluorescent carbon dots on fresh folium mori, feeding 5 instar larva of silkworms with the folium mori, and feeding the composite fluorescent carbon dots into silk glands of the silkworms by virtue of absorption and conversion of the silkworms selves; and (3) collecting fluorescent silk, namely continuously feeding the silkworms with the folium mori with the fluorescent carbon dots till the silkworms spin and cocoon, and collecting silkworm cocoons after cocooning, thereby obtaining fluorescent silk. By adopting the method disclosed by the invention, no dip dyeing step is implemented in the whole process, no adhesive is used, tedious steps of water washing, drying and the like are avoided, under the condition that the natural quality of silk is not damaged, green preparation of the fluorescent silk is achieved, and silk with fluorescent properties can be made.

The present invention addresses the problem of providing: a posterior silk gland gene expression unit, whereby a recombinant peptide, in particular a water-soluble peptide, can be expressed in a large amount in the posterior silk gland of a silkworm such as Bombyx mori; and a transgenic silkworm into which the aforesaid gene expression unit has been transferred. Provided is a posterior silk gland gene expression unit which comprises: a promoter of a gene encoding a protein, said protein being expressed specifically in the posterior silk gland; a DNA encoding a signal peptide originating in a protein, said protein being expressed specifically in the middle silk gland, functionally bound to the downstream of the promoter; and a DNA encoding a target peptide, said peptide being free from proteins secreted as constituents of fibroin, ligated to the downstream thereof.

The invention discloses a method for preparing high-thermal-conductivity silk by feeding silkworms with nano graphene and a product of the silk, and belongs to the fields of silk fibers and modification thereof. The method specifically comprises the steps that mulberry leaves are evenly coated with the nano graphene, the nano graphene is made to enter silk glands of the silkworms by utilizing theabsorption conversion function of the silkworms, and the high-thermal-conductivity silk containing the nano graphene is obtained through self-cocooning of the silkworms. On the premise that the natural quality of the silk is not damaged, the silk is modified, and the obtained silk has good thermal conductivity.

The invention discloses a method for increasing weight of Bombyx mori pupa bodies. The method includes the following steps: extracting total RNA from fifth instar Bombyx mori silk glands; performing reverse transcription to obtain first-chain cDNA, and performing Rt-PCR amplification by taking the first-chain cDNA as a template; obtaining BmCrzR cDNA through cloning; performing PCR amplification by taking BmCrzR cDNA as a template; after enzyme digestion, connecting DNA segments with a carrier to obtain pFLAG-BmCrzR; performing PCR amplification by taking pFLAG-BmCrzR as a template to obtain a reaction template; mixing the reaction template with T7 reaction buffer, ATP liquid, CTP liquid, GTP liquid, UTP liquid and T7 enzyme for reaction, and obtaining BmCrzR dsRNA after further treatment; finally, injecting BmCrzR dsRNA into abdomens of larvae of fifth instar Bombyx mori; feeding with folium mori until Bombyx mori are mature and cocooning is formed and weight of Bombyx mori pupa bodies is increased. The method for increasing weight of Bombyx mori pupa bodies is realized through controlling Bombyx mori metabolism rather than increasing eating quantity of folium mori, so that the method has positive functions on studying the substance and energy metabolism mechanism of Bombyx mori and further improving the physiological function of Bombyx mori.

The present invention discloses a method which utilizes the in-vitro expression system of the posterior silkgland of Bombyx mori to synthesize a protein. pUC19-FL-GFP-PolyA recombinant plasmid or pUC19-FL-GFP-PolyA recombinant plasmid with an exogenous gene or a pUC19-FL-PolyA recombinant plasmid or a pUC19-FL-PolyA recombinant plasmid with an exogenous gene and protease inhibitor are added into the tissue extract of the posterior silkgland of Bombyx mori, and after water bathing under the temperature between 25 DEG C and 29 DEG C, the expression of the targeted protein is obtained. The method has the following advantages: since the protein is expressed under a temperature between 25 DEG C and 29 DEG C, the low-temperature condition (normally, 37 DEG C) helps to generate the expressed protein in a dissolved state, and the formation of inclusion bodies is reduced, which is favorable for the separation and purification of the protein. Meanwhile, the method is not limited by cells and can synthesize the protein in a short period.