58 results about "Triple helix" patented technology

The invention relates to a recombinant I-type humanized collagen with crosslinking property as well as a preparation method and application thereof, and belongs to the technical field of recombinant proteins. The invention relates to recombinant I-type humanized collagen with crosslinking property. The amino acid sequence of the recombinant I-type humanized collagen comprises an amino acid sequence as shown in SEQ ID No.1 or a derivative amino acid sequence with the sequence identity of more than 90% with the sequence as shown in SEQ ID No.1; the recombinant I-type humanized collagen has a triple helix structure. A complete triple helix structure can be efficiently expressed, and the hydrogel has efficient cross-linking capability, so that the hydrogel can be efficiently and stably formed through cross-linking.

The present invention relates to the technical field of protein engineering and provides a recombinant humanized type III collagen having a triple helix structure, a preparation method therefor, and a use thereof. The amino acid sequence of the recombinant humanized type III collagen comprises n core units, wherein n is 1 or an integer greater than 1, and the amino acid sequence of the core unit is as shown in SEQ ID NO: 1. The present invention further provides a method for constructing an expression system and a method for preparing a recombinant humanized type III collagen. The present invention further provides an actual industrial use on the basis of the characteristics of the protein. The recombinant humanized type III collagen provided by the present invention has high purity, good stability, and high biological activity. Experiments have shown that the recombinant humanized type III collagen has various biological activities such as promoting cell proliferation and migration.

The invention relates to the technical field of synthetic biology, in particular to polypeptide with a triple helix structure, recombinant XII type humanized collagen and application of the recombinant XII type humanized collagen. The recombinant XII type humanized collagen is successfully expressed and prepared, has a triple-helix structure and good biological activity including promotion of cell proliferation activity, cell adhesion activity and inhibition of MMP-1, is used as a biological material derived from a human body, does not generate immunological rejection and anaphylactic reaction when applied to the human body, and has a good application prospect. The composition can be used for medical or non-medical application of a plurality of tissues and organs of a human body, filling, compatibilizing or repairing, and promotion of skin compactness or wrinkle resistance and other scenes.

The application discloses a recombinant collagen type XVII and a preparation method thereof, and relates to the technical field of genetic engineering. The application provides a recombinant collagen type XVII. Compared with the prior art, the recombinant collagen type XVII provided by the application has high yield of Pichia pastoris engineering bacteria, is not easy to be degraded, and has a stable triple helix structure.

The present disclosure provides recombinant type XVII collagen having a triple helix structure and uses thereof, which belong to the field of protein engineering technology. The recombinant type XVII collagen has an amino acid sequence formed by tandem repeats of n or more core units, where n is an integer of 1 or more, and the core unit has the amino acid sequence shown in SEQ ID NO: 1. The present disclosure also provides methods for constructing multiple expression systems and methods for preparing recombinant type XVII collagen. provide.

The invention provides recombinant II-type collagen as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. The recombinant II-type collagen monomer is obtained by splicing Gly-X-Y repetitive gene sequences of multiple sections of triple helix regions in human II-type collagen, immunodominant epitopes, unstable amino acid sequence 3 conjunctions and glycosylation sites are avoided during selection, then the recombinant II-type collagen monomer is repeated for multiple times, and the recombinant II-type collagen monomer is obtained. The recombinant II type collagen with the molecular weight of 40-50 kDa is obtained; the recombinant II-type collagen is beneficial to expression of pichia pastoris, has the effects of promoting adhesion and migration of cartilage cells, can also promote differentiation into cartilage cells, and has good practicability.

This invention relates to the field of biomedical materials, providing non-denatured type II collagen ultrafine powder with cartilage tissue-targeted growth and structural repair functions, its bioactivity preservation preparation method, and its application in joints. The ultrafine powder has an average particle size of 0.5-50 μm, a triple helix structure integrity rate of not less than 85%, a thermal denaturation temperature of 38-48℃, and retains chondrocyte binding domains CB10, CB11, and MMP cleavage sites. The preparation method includes raw material pretreatment, time-limited enzymatic hydrolysis, graded purification, and ultra-low temperature ball milling. The product can be used for the repair and treatment of cartilage degenerative diseases such as osteoarthritis and cartilage damage.

The invention discloses a polypeptide, a recombinant XI type humanized collagen formed by the polypeptide and application of the recombinant XI type humanized collagen. Through a large number of screening studies, it is found that a natural human XI type collagen core functional region of recombinant XI type humanized collagen with a triple helix structure can be formed in the in-vitro biosynthesis process. On the basis, the recombinant humanized polypeptide is designed and prepared on the basis of a natural XI type collagen core functional area, and the polypeptide can form a stable triple-helix structure. The recombinant XI type humanized collagen provided by the invention has good biological activities, including promotion of cell proliferation activity, promotion of cell adhesion activity and promotion of extracellular matrix generation or reconstruction, and is suitable for various medical or non-medical applications.

The present disclosure provides a recombinant humanized collagen VIII, which is a triple helix structure, has good cell adhesion activity, and is applied to human body without immune rejection and allergic reaction.

This invention discloses a method for maintaining the triple helix structure during the preparation of methacrylated recombinant collagen hydrogels, belonging to the field of genetic engineering technology. This invention optimizes the reaction parameters of the methacrylic anhydride (MA) chemical cross-linking process of recombinant collagen. Through screening, the titration method with an addition rate of 2 μL / min for MA was selected. The reaction temperature and the amount of MA added were optimized, and it was found that a MA:Col ratio of 1:2 and a reaction temperature of 35℃ resulted in good triple helix maintenance. This invention, through the titration method of adding methacrylic anhydride, greatly maintains the triple helix structure of recombinant collagen during the preparation of hydrogels, preserving excellent biocompatibility and showing broad application prospects in the field of biomaterials.

A self-crosslinking recombinant humanized collagen polymer biomaterial and a preparation method therefor are provided. A collagen self-assembled element includes (i) at least 21 consecutive amino acids at the C-terminus of the amino acid sequence as shown in SEQ ID NO: 1; or an amino acid sequence that is obtained by means of substitution, deletion or addition of one or more amino acids in the amino acid sequence as described in (i) and that retains the function of promoting the self-crosslinking of recombinant type III humanized collagen to form a triple helix structure; or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as described in (i) and that retains the function of promoting the self-crosslinking of recombinant type III humanized collagen to form a triple helix structure.

A new preparation method of recombinant humanized collagen hydrogel is provided. The method comprises the following steps: reacting recombinant humanized collagen and a dialdehyde cross-linking agent, wherein the concentration of the dialdehyde cross-linking agent is 0.01-0.25%, and the recombinant humanized collagen is one or more of recombinant humanized type II, type III and type XVII collagen, and the recombinant humanized collagen has a triple helix structure. The present application determines the aldehyde cross-linking agent suitable for recombinant humanized collagen, especially the recombinant humanized collagen with a triple helix structure and the optimum reaction conditions.

This invention relates to bioengineering, specifically disclosing a method for preparing recombinant humanized type III collagen with a triple-helix structure, comprising the following steps: S1, constructing a ppICZαA recombinant expression plasmid containing a human type III collagen fragment, and transforming the recombinant expression plasmid into yeast; S2, screening high-copy transformant strains from the strain and performing shake-flask level induction expression tests; S3, conducting high-density fermentation in a fermenter to obtain a large amount of secretible target protein. This invention, by constructing a ppICZαA recombinant expression plasmid containing a human type III collagen fragment and electroporating it into a yeast expression system, screens for yeast strains capable of expressing recombinant humanized type III collagen fragments, and conducts high-density fermentation in a fermenter, followed by purification after fermentation, yields pure recombinant collagen with a triple-helix structure, which can be applied in skincare products, functional skincare products, medical devices, and biomedical materials.

Provided is a method for preparing a recombinant human collagen. The method comprises: constructing a genetically engineered bacterium which expresses a prolyl hydroxylase and a recombinant human collagen; culturing the genetically engineered bacterium and performing digestion treatment by means of pepsin to obtain a recombinant human collagen type I or type III. The prolyl hydroxylase comprises P4Ha2, P4Hb and vP4H, and by means of controlling the ratio of the copy numbers of the genes thereof, the recombinant human collagen and a natural human collagen are consistent in hydroxylation modification. Also provided is a large-scale production method for the recombinant human collagen. The prepared recombinant human collagen has completely the same sequence composition and length and substantially the same hydroxyproline content as the natural human collagen, and also has a stable full-length triple helix structure and biological activity without immunogenicity and potential risk of viruses.

The invention provides a recombinant I-type collagen with a triple helix structure and application thereof, and belongs to the technical field of protein engineering, the amino acid sequence of the recombinant I-type collagen comprises n repetitive units, n is an integer of 1 or greater than 1, and the amino acid sequences of the repetitive units are as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7; the invention also provides a construction method of the expression system and a preparation method of the recombinant I-type collagen. The invention also provides practical industrial application according to the characteristics of the protein. The recombinant I-type collagen provided by the invention is high in purity and has a stable triple-helix structure.

The invention belongs to the technical field of synthetic biology, and particularly relates to a polypeptide, a recombinant XI-type humanized collagen composed of the polypeptide and application of the recombinant XI-type humanized collagen. According to the invention, through a large number of screening researches, a core functional region of a natural human XI type collagen alpha1 (XI) chain and a natural human XI type collagen alpha2 (XI) chain of a recombinant XI type humanized collagen with a triple helix structure can be formed in an in-vitro biosynthesis process. Furthermore, the polypeptide capable of forming the recombinant XI type humanized collagen with the triple-helix structure and the recombinant XI type humanized collagen with the triple-helix structure based on the polypeptide are successfully expressed and prepared for the first time. The recombinant XI type humanized collagen provided by the invention has good biological activity, including promotion of cell proliferation activity and promotion of cell adhesion activity, and is suitable for various medical or non-medical applications.

The invention belongs to the field of bioengineering, and discloses a soluble humanized III-type collagen with a stable triple helix structure, and a preparation method and application thereof. According to the invention, a bioinformatics method is adopted to preferably splice functional fragments from a humanized III-type collagen sequence, and a coding gene is integrated into a pichia pastoris genome in a multi-copy form to obtain a high-expression humanized III-type collagen strain; high-expression target protein is obtained through biological fermentation, the target protein with the purity of 95% or above can be obtained through one-step chromatography, the production efficiency is obviously improved, the process is simple and easy to operate, and the method is suitable for industrial amplification. The prepared humanized III-type collagen has a triple helix structure, is stable in structure, has the activities of promoting cell migration, promoting cell proliferation, resisting inflammation and the like, and has great value in the fields of medical beauty and medicine.

The present application relates to a kind of recombinant human type III collagen and its preparation method and application, belong to the field of bioengineering technology.The amino acid sequence of the recombinant human type III collagen described in the present application is as shown in SEQ ID No.7, is through the directional arrangement of five characteristic triple helix domain and the expansion of polymerization, combined with high-density fermentation process of Pichia pastoris, successfully break through the technical barrier of 4.5g / L target protein concentration.The present application research shows that the recombinant protein at 0.5% concentration not only maintains more than 80% cell viability, more exhibits significant synergistic effect-promote fibroblast migration rate to increase more than 29% compared with blank control group, keratinocyte proliferation rate increases 40.62%, adhesion rate increases 6.53%, provides innovative solution for developing new collagen product with high-efficiency expression and multiple biological activities.

The invention discloses low-immunogenicity, high-purity and high-bioactivity collagen as well as a preparation method and application thereof, and belongs to the technical field of medical material preparation. According to the preparation method disclosed by the invention, transgenic pigskin is taken as a raw material, enzymolysis is carried out under monitoring of an intelligent system, and collagen with low immunogenicity, high purity and high biological activity is prepared by combining salting-out with an isoelectric point separation technology and a multi-stage purification technology. And low immunogenicity, high purity and high biological activity of the collagen can be realized while the complete triple-helix structure of the collagen is ensured. The prepared collagen can be applied to the fields of medical treatment and medical beauty.

The invention provides a method for preparing recombinant I-type human collagen. The method comprises the following steps: carrying out fermentation culture treatment on engineering bacteria, wherein the engineering bacteria express precollagen; carrying out purification treatment on the fermentation culture treatment product so as to obtain the recombinant I-type human collagen; wherein the fermentation culture treatment is three-stage fermentation, and the final fermentation volume of the three-stage fermentation is not less than 9 tons. According to the method, the recombinant I-type human collagen with a complete triple helix structure, low immunogenicity, high safety and high purity can be prepared on a large scale, the recombinant I-type human collagen is composed of two alpha1 chains and one alpha2 chain, the hydroxyproline content is high, and the product stability is good.

The application provides a recombinant collagen XVII fragment, and the protein fragment sequence is shown as SEQ ID No. 1. The recombinant collagen XVII fragment is designed based on a natural collagen sequence, has a triple helix structure, and has good biological activity, and can effectively play a function, and has a good promoting effect on cell migration, cell proliferation and cell adhesion. The method for preparing the recombinant collagen XVII fragment has simplified process steps, improves the expression efficiency of the recombinant protein, and reduces the production cost. The recombinant collagen XVII fragment has good mechanical properties and biological activity, and can be used for preparing a therapeutic drug and a biological material.

The invention relates to a crosslinkable recombinant III-type humanized collagen as well as a preparation method and application thereof, and belongs to the technical field of recombinant proteins. The invention relates to a crosslinkable recombinant III-type humanized collagen. The amino acid sequence of the recombinant III-type humanized collagen is (GXY) n; wherein n is greater than or equal to 1; the GXY comprises an amino acid sequence as shown in SEQ ID No.1 or a derivative amino acid sequence, wherein the sequence identity of the derivative amino acid sequence and the SEQ ID No.1 is 90% or above; and the recombinant III type humanized collagen has a triple helix structure. On the basis of low immunogenicity, the hydrogel has a relatively stable triple helix structure, and has excellent cross-linking property, so that the hydrogel is efficiently and stably formed through cross-linking.

The invention discloses recombinant III-type collagen with thermal stability as well as a preparation method and application thereof, and belongs to the field of medical materials. The amino acid sequence of the recombinant III type collagen with thermal stability is any one of SEQ ID NO: 4-6. Based on rational design and functional optimization of the amino acid sequence, a key active sequence and a triple helix structure which are consistent with those of natural human body collagen are completely reserved; meanwhile, efficient and controllable large-scale preparation by utilizing an escherichia coli expression system is realized. The recombinant III-type collagen obtained by the invention shows excellent thermal stability, and a stable, reliable, economical and feasible raw material solution meeting strict quality control requirements is provided for large-scale and industrial application of the collagen in the fields of medical instruments, functional skin care products, tissue engineering and the like.

The invention provides a purification method of recombinant collagen, which comprises the following steps: taking thalli containing recombinant collagen, and carrying out resuspension, crushing and centrifugation to obtain a first supernatant; adding a viscosity reducer into the obtained first supernate, and centrifuging to obtain second supernate; and carrying out metal chelating affinity chromatography on the obtained second supernatant, and collecting an elution peak solution to obtain the product. According to the method for purifying the recombinant collagen, the obtained recombinant collagen is easy to form a stable triple helix structure and has better biological activity.

The application provides a kind of recombinant human collagen XVII and its preparation method and application, it is related to genetic engineering technical field.The preparation method of recombinant human collagen XVII includes the following steps: selecting the amino acid sequence Xvii-rich of GPP of human collagen XVII triple helix region 1127 to 1149 position, then codon optimization of yeast expression system is carried out, and the recombinant human collagen XVII is prepared according to the sequence of target gene.The molecular weight of recombinant collagen XVII is reduced compared with human collagen XVII, is not easy to degrade, and is 100% homologous with the amino acid sequence of natural collagen XVII, has no immunogenicity;By electrophoresis detection, the proportion of the complete target protein of recombinant human collagen XVII secreted to the outside of host cell is more than 90%, has the advantages of high stability and not easy to degrade.

The invention relates to the technical field of genetic engineering, in particular to recombinant I-type human collagen as well as a preparation method and application thereof. The invention provides recombinant I-type human collagen. The recombinant I-type human collagen comprises an amino acid sequence as shown in SEQ ID NO.7 or an amino acid sequence having identity with the amino acid sequence as shown in SEQ ID NO.7 according to a sequence from an N terminal to a C terminal. The recombinant I-type human collagen disclosed by the invention is optimized and modified by taking natural I-type human collagen as an original gene sequence. The sequence has a triple-helix structure characterized by collagen, a (GlyXY) n repeated amino acid sequence, a high proline region and an RGD repeated sequence, and can be self-assembled to form good triple-helix high-stability collagenous fiber; meanwhile, the preparation process is simple, and high-yield collagen can be obtained at low cost.

The application discloses a preparation and separation and purification method of deer antler peptide by directional enzymolysis, and belongs to the technical field of bioactive peptide preparation. Deer antler collagen is subjected to mechanical energy auxiliary treatment in an alkaline medium under low-temperature conditions lower than the thermal denaturation temperature of the deer antler collagen, so that the triple helix structure is reversibly unfolded to increase the accessible surface area of the protease and the conformation of the active segment of the ginseng protein is retained. The treated deer antler collagen and the ginseng protein are prepared into the same substrate solution and subjected to synergistic enzymolysis. Alkaline protease, collagenase and aminopeptidase are used for directional enzymolysis in steps, so that the product is directionally enriched with two characteristic dipeptides, prolyl-hydroxyproline and hydroxyprolyl-glycine. The dipeptides are separated by three steps of ultrafiltration, gradient elution of a macroporous resin and reverse phase preparative chromatography in series, so that the low-temperature mechanical energy reversible unfolding replaces the high-temperature pre-denaturation, the contradiction between the two types of raw materials in the treatment temperature window is eliminated, the two types of raw materials can be synergistically treated in the same system, and the dipeptide yield is higher than that of the separate enzymolysis.

This invention belongs to the field of biomedical material preparation technology, specifically disclosing a method for moist heat sterilization of recombinant collagen fillers. The method includes: preparing a protective solution from modified trehalose and proline, then mixing it with homogenized recombinant collagen hydrogel, followed by staged temperature-controlled moist heat sterilization. This invention modifies trehalose by succinylation, introducing carboxyl groups onto the trehalose molecule, enabling electrostatic interactions with cationic groups on the surface of collagen molecules. During moist heat sterilization, free proline guides the heat-denatured collagen to refold into its natural triple helix structure. The preheating stage allows the collagen to gradually adapt to the thermal environment, avoiding conformational shock damage caused by sudden temperature increases, effectively shortening the high-temperature sterilization time, and significantly reducing the cumulative heat effect on molecular structure. This method is simple to operate, requires no special equipment, and has good prospects for industrial application.

The application belongs to the technical field of biological medicine, and particularly relates to a recombinant collagen repair solution and a preparation method and application thereof. The application comprises the following steps: preparation of a protein activity enhancer; preparation of a recombinant humanized collagen fiber sol; and photo-polymerization construction of a recombinant humanized collagen hydrogel. The application is modified by sulfate esterification of pollen of apiaceae polysaccharide, and is compounded with the recombinant humanized collagen. The application can not only form a stable complex with the positively charged collagen amino acid residues through electrostatic interaction, maintain the natural triple helix conformation, reduce the aggregation and denaturation of the collagen, and enhance the activity of the collagen, but also can assist the recombinant humanized collagen in activating the cell surface receptors, enhancing the adhesion and proliferation of the cells, thereby enhancing the epidermal regeneration capacity of the recombinant humanized collagen, effectively improving the bioavailability of the recombinant humanized collagen at the wound site, and improving the repair and treatment effect on the wounded skin.

This invention discloses a method and application for constructing transgenic silkworm lines that enhance the triple-helix structure of recombinant full-length human type III collagen using a binary hybridization strategy. First, this invention constructs a transgenic silkworm line co-expressing human prolyl 4-hydroxylase (hP4H) α and β subunits, and a transgenic silkworm line expressing recombinant full-length human type III collagen (rhCOLⅢ). Then, the two lines are genetically hybridized using a binary hybridization strategy, and double-positive offspring co-expressing rhCOLⅢ and hP4H are selected, achieving post-translational hydroxylation modification of rhCOLⅢ by hP4H. The modified recombinant full-length human type III collagen (rhCOLⅢ-Hyp) triple-helix structure content is significantly increased. This invention provides a key modification tool and technical pathway for producing full-length recombinant human collagen with a complete triple-helix structure using a silkworm bioreactor.