27 results about "Silk gland" patented technology

The invention discloses a silk material with a function of promoting cell proliferation and application of the silk material. According to the functional material, a human-derived I-type collagen truncated fragment is expressed by a silkworm middle silk gland, and a sericin promoter serving as a tissue-specific promoter can drive an exogenous gene to be specifically expressed in the middle silk gland; finally, the silkworm cocoons are combined with sericin to be secreted in vitro, foreign protein can be separated after certain treatment is carried out on the silkworm cocoons, and the expression product can maintain biological activity. The hydrogel prepared from the silk material has the effect of promoting wound healing, and can be applied to the aspects of promoting skin regeneration and repair, bone transplantation substitutes, cartilage reconstruction, artificial skin and the like.

The invention belongs to the technical field of biology, particularly discloses a method for preparing spider silk protein-containing composite silk by using transgenic silkworms and application of the spider silk protein-containing composite silk, and particularly provides a method for synthesizing and secreting spider silk protein by using a silkworm silk gland bioreactor. The spider silk protein gene is introduced into a bombyx mori genome in a virus infection manner by utilizing a transgenic bombyx mori technology and is specifically expressed in bombyx mori silk gland cells, and the spider silk protein target gene can be introduced in batches by virtue of a lentivirus transfection method, so that bombyx mori capable of synthesizing and secreting complete spider silk protein can be developed on a large scale; the spider silk protein is synthesized and secreted through bombyx mori silk gland cells and enters silkworm cocoons along with the spinning and cocooning behaviors of bombyx mori, the silk is collected, the spider silk protein-containing composite silk with the bombyx mori silk as the background is obtained, a foundation is laid for mass production of black spider silk protein, and meanwhile a foundation is laid for improving the mechanical performance of the silk.

The invention relates to the field of gene engineering, and discloses dsRNA of a bombyx mori glucose regulatory protein 78 gene and a preparation method of the dsRNA, and the preparation method comprises the following steps: a) designing and synthesizing an upstream primer and a downstream primer of a BmGRP78 gene; b) extracting bombyx mori total RNA, performing reverse transcription to obtain first chain cDNA, taking the first chain cDNA as a template, performing PCR amplification to obtain a BmGRP78 gene full-length fragment, purifying, cloning to escherichia coli, and performing sequencing verification; c) designing an upstream primer SEQ ID NO: 3 and a downstream primer SEQ ID NO: 4 of dsRNA according to the nucleotide sequence SEQ ID NO: 1 of the BmGRP78 gene, wherein both the upstream primer and the downstream primer carry a T7 promoter sequence; and d) in-vitro transcription synthesis of dsRNA which can specifically inhibit BmGRP78 gene expression and is used for bombyx mori to optimize silk production and improve disease resistance innovation. Compared with the prior art, the dsRNA has the effect of outstanding gene knock-down efficiency, and experimental results prove that the dsRNA can effectively reduce BmGRP78 gene expression in bombyx mori ovary cells (BmN) and silk glands.

The application discloses a silk material with a cell proliferation function and application thereof, the functional material is expressed by a human type I collagen truncated fragment in a middle silk gland of a domestic silkworm, a sericin promoter is used as a tissue-specific promoter to drive specific expression of an exogenous gene in the middle silk gland, and finally the exogenous gene is combined with sericin protein and is secreted to outside of the body, the exogenous protein can be separated after a certain treatment of a cocoon, and the expression product can keep biological activity. The silk material is used to prepare a hydrogel, has a function of promoting wound healing, and can be applied to aspects of promoting skin regeneration and repair, bone graft substitutes, cartilage reconstruction, artificial skin and the like.

The present invention discloses a silk improved by spider silk protein gene and a method for improving silk by spider silk protein gene, wherein the silk improved by spider silk protein gene comprises a silk protein light chain, and the silk protein light chain is connected with a spider silk protein formed by fusion expression with the silk protein light chain; the amino acid sequence of the spider silk protein comprises a repeating region sequence having at least one spider silk protein repeating unit sequence. The present invention realizes the expression of the pear-shaped gland silk of Astilbe punctata in Bombyx mori silk fibroin, realizes the formation of a complete spider silk protein on the silk light chain, and expands the gene editing site of the silk gland of Bombyx mori. This light chain editing silkworm can be crossbred with the heavy chain gene editing silkworm, providing technical support for further breeding and optimization of fiber performance.

The invention discloses a viscous tussah silk gland protein gel dressing with microenvironment response and antibacterial property and a preparation method thereof, N-isopropylacrylamide is polymerized to form poly-N-isopropylacrylamide through in-situ free radical polymerization, and meanwhile, the poly-N-isopropylacrylamide, tussah silk gland protein and sodium alginate form gel in a stable form through intermolecular interaction; on one hand, the gel dressing has good temperature-sensitive response performance and realizes intelligent drug controlled release, and on the other hand, the hydrogel can be well adhered to the skin surface, can promote growth and reproduction of human skin fibroblasts, shows good biocompatibility and mechanical properties, has better adhesion stress than commercial wound dressings, and has good application prospects. An application basis is provided for the application of the sodium alginate group in the intelligent medicine controlled release medical wound dressing; finally, the gel dressing is loaded with a natural antibacterial agent and Chinese herbal medicines, and has the effects of resisting bacteria, diminishing inflammation and the like at the affected part; therefore, the gel dressing disclosed by the invention has great application potential in the medical field.

The invention discloses a preparation method of active recombinant human adiponectin hydrogel as well as a product and application thereof. RhAPN (recombinant human adiponectin) gene as shown in SEQ ID NO.2 is connected to a Ser1 promoter to be expressed in silk glands of bombyx mori under regulation and control, transgenic individuals are screened and fed until cocooning, and silk containing recombinant human adiponectin is obtained; the prepared hydrogel can reduce lipid, can effectively treat mouse ulcerative colitis, and can be used for treating colitis.

The invention provides a method for separating complete intestinal tracts of greater wax moth larvae, which comprises the following steps of: 1, soaking the to-be-dissected greater wax moth larvae in alcohol, and disinfecting body surfaces; 2, taking the greater wax moth treated in the step 1, placing the greater wax moth in the center of a wax disc with the head being front and the tail being back and the back being upward, and nailing insect needles at the head and the tail; 3, making a transverse small cut on the back of the tail end, and cutting to the head end along the back center line; 4, opening the body wall and fixing by using an insect needle; and 5, adding dissecting operation liquid into the wax plate, taking down the whole digestive tract by using tweezers, and cleaning fat, silk glands and muscles. According to the method, under the condition that epidermis and other tissues of greater wax moth are not damaged, the tail of the greater wax moth is taken as a breakthrough opening, the acting point is found, and the complete intestinal tissues of the greater wax moth larva are rapidly drawn out, so that the integrity of the intestinal tract of the larva can be ensured to the maximum extent, and leakage of intestinal contents and pollution of other tissues in the body caused by mechanical injury are avoided. The dissecting time is greatly shortened, pollution and waste of materials can be reduced, and tissue degradation is avoided.

The invention discloses an expression method of a non-proteolytic thrombin peptide TP508 in bombyx mori silk gland and a product and application thereof, a fusion expression vector of the non-proteolytic thrombin peptide TP508 and a red fluorescent protein DsRED is constructed, the fusion protein is expressed in silkworm cocoon silk, the bred transgenic strain silk successfully expresses the fusion protein, and the non-proteolytic thrombin peptide TP508 is obtained. The thrombin peptide TP508 fusion protein expressed by the transgenic silk not only can remarkably promote cell proliferation, cell migration and vascularization, but also keeps the characteristic of red fluorescence, and is expected to be applied to the fields of biomedicine and tissue cell engineering.

The invention discloses an anti-yellowing silk, belongs to the field of silk fibers and modification thereof, and is a silk formed by spinning, cocooning and solidifying bombyx mori bred by nano silicon dioxide with amphiphilic properties and mulberry leaves. According to the method disclosed by the invention, the silk is modified on the premise of keeping good natural performance of the silk, and a scanning electron microscope and an inductively coupled plasma mass spectrometry prove that the nano silicon dioxide is embedded into the modified silk through midguts, blood and silk glands of silkworms, so that the mechanical property of the silk is obviously improved; the performance of the silk can be kept under high ultraviolet irradiation intensity, and the silk is effectively prevented from yellowing and embrittling. The method disclosed by the invention is green and environment-friendly, the prepared silk is safe and non-toxic, the anti-ultraviolet performance of the silk is obviously superior to that of common silk, the yellowing and embrittlement of the silk can be effectively prevented, and the silk has a wide application prospect in the fields of daily application, protection, biological materials and the like.

The invention discloses a construction method of bombyx mori for producing recombinant human III-type collagen and application of the bombyx mori, and belongs to the technical field of biology. On the basis of a bombyx mori silk gland bioreactor for efficiently synthesizing foreign proteins, specific expression of partial or complete full-length recombinant human III-type collagen in bombyx mori posterior silk glands is realized through a piggyBac mediated transgenic technology, and the recombinant human III-type collagen is successfully secreted into silkworm cocoons. The recombinant human III-type collagen which is single in component and high in safety is obtained through subsequent extraction and purification of posterior silk glands and silkworm cocoons, a novel biological manufacturing method of high-added-value protein is constructed, and meanwhile, the economic value of silkworms is greatly improved.

The application discloses application of a silkworm silk gland BmSPI45 protein in preparation of a product resisting red hair moss, and relates to the technical field of bioengineering, and has the technical scheme as follows: the application of the silkworm silk gland BmSPI45 protein in preparation of the product resisting red hair moss, and the amino acid sequence of the silkworm silk gland BmSPI45 protein is SEQ ID NO:1.The application uses an insect baculovirus expression system to perform in-vitro expression on a BmSPI45 recombinant protein, and obtains a recombinant pFastBac-BmSPI45 eukaryotic expression vector.The constructed pFastBac-BmSPI45 eukaryotic expression vector is transformed into a DH10Bac competent cell, and a recombinant Bacmid is extracted and obtained, the recombinant Bacmid is transfected, and the BmSPI45 protein is massively expressed.The BmSPI45 recombinant protein is subjected to antibacterial function research, and the result shows that the protein has the effect of resisting red hair moss, thereby providing a new protein alternative for development of an antibacterial preparation.

The application discloses application of specific knockout of a P53 gene in improvement of silk yield and a method thereof, and promotes development of a silkworm silk gland, increases expression of a silk protein gene and improves silk yield by specifically knocking out the P53 gene in the rear part of the silkworm silk gland through a CRISPR / Cas9 gene editing system, so as to provide a new breakthrough point and target for silkworm variety improvement aiming at improving a silk protein synthesis level and silk yield, and has a good application prospect.

The invention discloses an environment-friendly multi-antibacterial tussah silk gland protein nanofiber and a preparation method and application thereof.The preparation method specifically comprises the steps that an antibacterial agent, Chinese herbal medicine and a photo-thermal agent are sequentially added into a spinning solution, and a nanofiber membrane loaded with the antibacterial agent, the Chinese herbal medicine and the photo-thermal agent is prepared through an electrostatic spinning method; and standing and drying the nanofiber membrane to obtain the environment-friendly multi-antibacterial tussah silk gland protein nanofiber. The antibacterial agent is one or more of chitin, chitosan, nano chitosan, aloe and antibacterial peptide; the Chinese herbal medicine is one or more of coptis chinensis, dandelion, wormwood, honeysuckle and isatis root; the photo-thermal agent is a substance capable of generating a local thermal effect under the irradiation of near-infrared light; and then preparing the tussah silk gland protein nanofiber wound dressing with multiple antibacterial health-care effects by adopting an electrostatic spinning technology, wherein the wound dressing has the characteristics of multiple antibacterial properties, good air permeability, high Chinese herbal medicine content, strong durability and the like.

The present application discloses silk improved by a spider silk protein gene and a method for using a spider silk protein gene to improve silk. The silk improved by the spider silk protein gene comprises a silk protein light chain. The silk protein light chain is linked to a spider silk protein which is formed by fusion expression with the silk fibroin light chain. An amino acid sequence of the spider silk protein comprises a repeat region sequence having at least one spider silk protein repeat unit sequence. The present application achieves the expression of Nephila pilipes pyriform silk in Bombyx mori silk fibroin, implements formation of an intact spider silk protein on the silk light chain, and expands sites for Bombyx mori silk gland gene editing. Light-chain gene edited Bombyx mori and a heavy-chain gene edited silkworm can be hybridized, thereby providing technical support for further selective breeding and optimization of fiber performance.

The invention discloses a method for producing an active rhBMP9 bone repair material based on a double-transgene bombyx mori silk gland biosynthesis system as well as a product and application of the active rhBMP9 bone repair material, and realizes cooperative expression of complex precursor protein Pro-mhBMP9 and precursor protein invertase hFurin in bombyx mori posterior silk glands. An hBMP9 / hFurin double-gene biosynthesis system capable of completing precursor protein shearing, maturation and functionalized silk material synthesis in vivo is established, the rhBMP9 / CaP / Sericin composite scaffold is further constructed by extracting silk protein of double-gene transformation bombyx mori, and the rhBMP9 / CaP / Sericin composite scaffold shows excellent bone repair and angiogenesis capacities in both the cell level and the animal level, and can be used for preparing a bone repair material. And a solid experimental and theoretical basis is provided for a functional protein-material-tissue regeneration integrated strategy based on a silk gland biosynthesis system.

This invention discloses a cell marker gene for maintaining the ion environment in silk-secreting insects. KCNQ Identification and application of marker genes for maintaining ion environment in silk-secreting insects KCNQ The gene is as shown in a) or b) below: a) the nucleotide sequence is as shown in SEQ ID No. 1; b) a protein encoding the amino acid sequence shown in SEQ ID No. 2, or a protein encoding a potassium channel activity obtained by substitution, deletion, and / or addition of one or more amino acid residues. This invention has discovered... KCNQ The present invention maintains cell-specific high expression in an ionic environment. KCNQ The gene is homologously conserved in various silk-secreting insects and is specifically expressed in the anterior silk gland. It can be used for the precise identification of anterior silk gland cell types in silk-secreting insects, the molecular improvement of silk fiber properties, and the development of multifunctional silk protein materials, which has important application value.

Disclosed is a silk gland recombinant expression vector of silkworm expressing human epidermal growth factor, a preparation method and use thereof. The recombinant expression vector is formed by fusing truncated silk fibroin light chain N-terminal domain, C-terminal domain, optimized human epidermal growth factor and silkworm nuclear polyhedrosis virus enhancer, and removes the redundant part of the extra domain of endogenous silk protein contained in the fusion protein to the maximum extent.

The invention discloses a bombyx mori silk gland sample preparation method suitable for space transcriptome sequencing, and belongs to the technical field of insect biology.The bombyx mori silk gland sample preparation method suitable for space transcriptome sequencing comprises the following steps that S1, silk gland dissection and tissue pretreatment are conducted; step S2, embedding the silk gland tissue; s3, freezing and slicing the silk gland tissue; s4, sample RNA quality inspection; step S5: tissue permeabilization; step S6, precisely splicing the pieces; and S7, capturing the chip. The silk gland frozen section prepared by the method and the section splicing technology meet the sample preparation requirements of space transcriptome sequencing, and can be used for analysis of cell space composition, gene expression characteristics, cell communication and the like. According to the method, the problems of difficulty in complete longitudinal slicing, organ size and chip capture adaptation and the like caused by heterogeneity of form and function partition and multiple buckling shapes of silkworm silk secreting organs are solved.

Disclosed is a silk gland recombinant expression vector of silkworm expressing human epidermal growth factor, a preparation method and use thereof. The recombinant expression vector is formed by fusing truncated silk fibroin light chain N-terminal domain, C-terminal domain, optimized human epidermal growth factor and silkworm nuclear polyhedrosis virus enhancer, and removes the redundant part of the extra domain of endogenous silk protein contained in the fusion protein to the maximum extent.

To provide a method and system for purifying natural protein raw material, capable of purifying natural protein raw material directly from a worm body without manual operation only by a chemical engineering process.SOLUTION: The purification method comprises: a pretreatment in which a worm body of a silk worm is pulverized to produce a treatment solution containing a silk gland having a first objective substance and a second objective substance; a first separation treatment in which a sediment mainly containing the silk gland is obtained from the pretreated treatment solution by specific gravity separation and each part of the worm body other than the silk gland is removed; a second separation treatment in which an aqueous solution obtained by eluting the second objective substance from the sediment obtained in the first separation treatment is obtained, and the contents of the silk gland mainly containing the first objective substance and the aqueous solution containing the second objective substance are separately obtained; a first objective substance recovery treatment in which the first objective substance is obtained from the contents of the silk gland mainly containing the first objective substance obtained in the second separation treatment; and a second objective substance recovery treatment in which the second objective substance is obtained from the aqueous solution containing the second objective substance obtained in the second separation treatment.SELECTED DRAWING: Figure 1

The invention discloses application of a bombyx mori silk gland peroxidase BmPOD gene in changing sericin viscidity and a method of the bombyx mori silk gland peroxidase BmPOD gene. Peroxidase BmPOD specifically expressed by a silk gland is knocked out through CRISPR / Cas9, a homozygous mutation individual is screened, complete knockout of peroxidase BmPOD in the silk gland is achieved, the viscidity of sericin in the silk gland of a bred gene knockout strain is greatly reduced, and the sericin viscidity of the bred gene knockout strain is improved. The degumming rate of silkworm cocoons is reduced, and the method is expected to be applied to production of sericin products with different characteristics or multi-sericin products.

The invention discloses a spider silk protein transgene production method and application based on lentiviral transfection and sperm mediation in the field of biotechnology and genetic engineering. The spider silk protein transgene production method comprises the following steps: firstly, constructing a vector pLC-[3xP3-DsRed]-MaSp2-Amyloids for synthesizing and secreting spider silk protein genes by bombyx mori; the plasmid is introduced into a silkworm fertilized egg by using a lentivirus transfection and sperm-mediated gene transfer method, the red fluorescent protein gene and the black widow spider dragline silk protein 2 and amyloid polypeptide composite gene are introduced into a silkworm genome by using the characteristics of the lentivirus, and stable inheritance and expression are obtained; therefore, a transgenic silkworm capable of specifically synthesizing and secreting spider silk protein in bombyx mori silk gland cells is created, then the spider silk protein is synthesized and secreted by the silkworm, and the spider silk protein-containing composite silk with bombyx mori silk as the background is obtained. The method lays a foundation for mass production of black spider silk protein and also lays a foundation for improvement of mechanical properties of silk fibers.

The invention discloses a preparation method of high-solubility mussel byssus gland mucoprotein for thickening a skin basal layer, which comprises the following steps: taking out common mussel byssus glands as raw materials, naturally unfreezing, cleaning, keeping the completeness of the byssus glands, carrying out double-enzyme treatment, sequentially carrying out double-enzyme enzymolysis by using neutral protease and pepsase, filtering, washing, and drying to obtain the high-solubility mussel byssus gland mucoprotein for thickening the skin basal layer. After the enzymolysis is finished, heating a reaction system, keeping the activity of the passivating enzyme, and removing an enzymolysis mixed solution; the preparation method comprises the following steps: washing byssus gland subjected to double-enzyme enzymolysis, adding a urea solution for homogenization treatment, removing insoluble impurities through efficient centrifugation, reserving a supernatant as a crude extract of byssus gland mucoprotein, carrying out microfiltration and nanofiltration through a ceramic membrane and a nanofiltration membrane, and carrying out spray drying or freeze drying on the collected liquid to obtain high-solubility mussel byssus gland mucoprotein powder. The high-solubility mussel byssus glandular mucoprotein is obtained through a double-enzyme urea nanofiltration engineering method, and the mussel byssus glandular mucoprotein has molecular uniqueness and high biological activity efficiency and is wide in application prospect.

This invention discloses the application of BmPriL in improving the economic traits of silkworms. Using the piggyBac transposition system, silkworms were constructed in which BmPriL was overexpressed specifically in the middle silk gland, mediated by the Ser1 promoter, and in the posterior silk gland, mediated by the FibH promoter. Results showed that the sericin content in cocoons of silkworms overexpressing BmPriL specifically in the middle silk gland was significantly higher than that in controls; and the cocoons overexpressing BmPriL specifically in the posterior silk gland were significantly higher in cocoons with significantly higher cocoon layer volume, cocoon layer rate, and fibroin content than in controls. These results demonstrate that BmPriL can improve the economic traits of silkworms.

The invention relates to the field of gene engineering, in particular to a recombinant vector of a bombyx mori silk gland expression human neurotrophic factor NT-3 as well as preparation and application of the recombinant vector. An optimized gene is constructed in a bombyx mori silk gland expression vector pBac [DsRed] plasmid by modifying a human-derived neurotrophic factor NT-3 gene, such as a sequence shown in SEQ ID NO 2, so that a pBac [DsRed, NT-3] recombinant plasmid is obtained, the recombinant plasmid and an auxiliary plasmid are mixed and microinjected into fresh silkworm eggs which are just laid by bombyx mori within 2 hours, a G0 generation is obtained, then a G1 generation is copulated and bred, and the G0 generation and the G1 generation are subjected to separation and purification. And screening out silkworms expressing the human neurotrophic factor NT-3 from the G1 generation. The optimized human neurotrophic factor NT-3 gene vector expressed in bombyx mori is constructed, the human neurotrophic factor NT-3 protein hydrogel is obtained, and the human neurotrophic factor NT-3 has potential application value in cell proliferation, cell migration and repair.