CN107988183A - A kind of recombined human hyaluronic acid and its production method - Google Patents

A kind of recombined human hyaluronic acid and its production method Download PDF

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CN107988183A
CN107988183A CN201711144684.2A CN201711144684A CN107988183A CN 107988183 A CN107988183 A CN 107988183A CN 201711144684 A CN201711144684 A CN 201711144684A CN 107988183 A CN107988183 A CN 107988183A
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hyaluronic acid
recombined human
hyaluronidase
human body
human
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贺晓军
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Hangzhou Huidoctor Biotechnology Co Ltd
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Hangzhou Huidoctor Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2474—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01035—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase

Abstract

The present invention provides a kind of as follows with the homologous recombined human hyaluronic acid of human body, the recombined human hyaluronic acid structural formula：Wherein n=3 10, structural formula both ends are and human body homology digestion end.Recombined human hyaluronic acid prepared by this method also has effects that inhibiting bacteria and diminishing inflammation, strengthens the human body activity of skin and mucosa immunity in addition to having the function of normal transparent matter acid.It can be widely used in the beauty and conditioning of the face in human body, oral cavity, hair, body and privates etc., make that safety coefficient higher, stability is more longlasting, the stronger daily chemical product of practicality.

Description

A kind of recombined human hyaluronic acid and its production method
Technical field
The present invention relates to filed of daily-use chemical industry, more particularly to a kind of recombined human hyaluronic acid and its production method.
Background technology
Hyaluronic acid, also known as Hyaluronic Acid, are a kind of high molecular polymer, by unit D-Glucose aldehydic acid and N- acetyl Portugal The advanced acid mucopolysaccharide of osamine composition, it is the tender important foundation material of skin water, itself is also a kind of component of human body, it With special water retention, deal is more up to 100 times of itself weight, is that moisture retention is best in presently found nature Material, it can also improve skin-nourishing metabolism, make skin it is tender, it is smooth, go to wrinkle, increase it is elastic, prevent aging, in moisturizing While be good skin penetration enhancer again, be used cooperatively with other nutritional ingredients, can play and promote nutrient absorption More preferable effect.In the source of existing hyaluronic acid, the method that animal is extracted is since raw material is limited, and hyaluronic acid in raw material Content is low, causes that yield is low, purification is difficult, of high cost, product quality is poor, it is difficult to large-scale production；The side of biofermentation extraction Method advantage is that yield is big, cost is low, occupies the 70% of world market, but the technique fermentation period used is often longer, product egg White matter content is high, glucuronic acid content is low, product light transmittance is poor, and the form of threadiness is even presented in some, not readily dissolves, The hyaluronan molecule amount obtained at the same time is larger, and generally between 80-120 ten thousand, major function is water conservation moisturizing and forms tissue Protective film and filler, can not be absorbed by human skin；At present using macromolecular hyaluronic acid as raw material, using machinery, chemistry, The Different treatments such as enzyme, the method for manufacturing low molecular weight hyaluronic acid are widely accepted due to overcoming disadvantage mentioned above, feature It is absorption easier than macromolecular hyaluronic acid, the function of hyaluronic acid can be produced after absorption, such as water lock moisturizing, optimization generation Thank to environment, promote Wound healing etc., but this kind of method obtains hyaluronic acid not only low output but also, institute active almost without human body The active efficacy effect of substance can not be played in the application to human body.
It is reported that hyaluronidase be degrade hyaluronic acid enzyme family, it can by be catalyzed the hydrolysis of hyaluronic acid come Length, molecular weight and the viscosity of hyaluronic acid are reduced, can quickly produce the hyaluronic acid fragments for meeting different demands.Root According to the mechanism of action and the difference of catabolite, general hyaluronidase can be divided into three classes, the first kind includes β-D- acetyl-D- Hexosaminidase, tetrose D-Glucose aldehyde is degraded into by high molecular weight substrate such as hyaluronic acid, chondroitin or chondroitin sulfate A (CSA) and C Acid-N-acetyl-glucosamine-D-Glucose aldehydic acid-N-acetyl-glucosamine is as main end-product, such as testicular hyaluronidase energy Transglycosylation is enough catalyzed, therefore hexose, disaccharides and eight carbon sugar can be also formed during hyaluronic acid is hydrolyzed；Second Class, the hyaluronidase using leech hyaluronidase as representative, by high molecular weight substrate hyaluronic acid degradation into tetrose N- acetyl Aminoglucose-D-Glucose aldehydic acid-N-acetyl-glucosamine-D-Glucose aldehydic acid is as main end-product；Three classes, bacterium are transparent Matter acid enzyme, it is a kind of lyases, and high molecular weight substrate hyaluronic acid or chondroitin are degraded into undersaturated N- Acetylglucos Amine-D-Glucose aldehydic acid polysaccharide structures are as main end-product.Research is found using big point general of hyaluronidase degraded Sub- hyaluronic acid obtain the method for micromolecule hyaluronic acid fragment because there are the homology of enzyme, the half-life period of enzyme, raw material sources and Enzymolysis process etc. influences the problem of Product yields and active product human body, and urgently further optimizes and solve, so finding one It is extremely urgent to plant the more effective method that can prepare the micromolecule hyaluronic acid with human body activity.
The content of the invention
In order to solve the above technical problems, what the present invention was realized in：
This method uses the recombined human hyaluronidase substitution homologous with human body obtained after transgenic technology optimization processing General hyaluronidase carries out digestion to the macromolecule hyaluronic acid of selection, obtains homology digestion end, and to digestion Technique is optimized and regulated and controled, and is obtained largely few comprising the hyaluronic acid of 3-10 dissacharide units, molecular weight in 1000-4000D Bglii fragment, the hyaluronic acid fragments comprising homology digestion end of these specific small molecule amounts are recombined human hyalomitome Acid, can obtain recombined human hyaluronate after further treatment.
(1) a kind of with the homologous recombined human hyaluronidase of human body, the recombined human hyaluronidase that the present invention uses is behaved The extracellular parts of hyaluronidase PH20 and the fusion protein of people's source protein (human albumin or immunoglobulin) fragment, its amino Acid sequence contains 644 amino acid residues, has the advantages that human body homology height, long half time.
Tangible 8 Cys into disulfide bond of recombined human hyaluronic acid enzyme molecule, 5 N- glycosylation sites from structure Asn-Ala-Thr, Asn-Arg-Ser, Asn-Gly-Ser, Asn-Glu-Ser, Asn-Val-Thr, 3 enzyme activity sites, these The presence of functional structure can improve the stability of recombined human hyaluronidase, extend half-life period, while can also increase restructuring Activity and the functionalization effect of people's hyaluronidase.Further study show that 4 disulfide bond of recombined human hyaluronic acid enzyme molecule Position is Cys25-Cys316、Cys189-Cys203、Cys341-Cys571、Cys429-Cys481, 5 N- glycosylation sites are Asn47- Ala48-Thr49、Asn131-Arg132-Ser133、Asn200-Gly201-Ser202、Asn219-Glu220-Ser221、Asn333-Val334- Thr335, 3 enzyme activity sites are Asp111、Glu113、Gln276.Activated centre, that is, substrate-binding site, rolls over positioned in C- ends and β In groove between folded, active group Asp111、Glu113The random ravel area in groove, substrate (D-Glucose aldehydic acid-N- Acetylglucosamine)3-10Polymer in groove, can obtain including 3-10 dissacharide units under the conditions of certain digestion just With the micromolecule hyaluronic acid oligose fragment of human body homology.Wherein Glu113For proton donor, and the N- acetyl group of hyaluronic acid Group is polarity nucleophile, both carry out digestion effect under close specific binding, form homology digestion end, enzyme activity base The position of group is very peculiar to be similar to bacterium chitinase.
(2) the homologous recombined human hyaluronidase preparation method of above-mentioned human body
A kind of and homologous recombined human hyaluronidase preparation method of human body, includes the following steps：
1) that the target gene of fusion protein recombined human hyaluronidase is building up to the animal rich in GC by PCR method is thin In cellular expression carrier pMH3, pMH4 or pMH5；
2) after preparing expression plasmid, it is stably transfected into the Chinese hamster ovary celI of serum free suspension culture；Screened, used by G418 The manual picking stable clone of pipette tips is into 96 orifice plates；
3) when cell confluency degree is more than 50%, serum-free fresh culture is replaced；
4) after when 3-6 is small, media samples are collected, expression quantity is detected by antibody dot blot or ELISA method, selects table Up to highest multiple clones are measured, continue to carry out free serum culture in the small shaking flask in sharp bottom and do mitotic stability research；
5) select the stable clone of passage and carry out amplification cultivation in 5-8L rip current type reactors, in each batch good harvest liquid The middle nutrient solution chosen hyaluronidase activity concentration and reach 600-1000U/L.
Preferably, the recombined human hyaluronidase preparation method homologous with human body, further includes after step 5) Following steps：
1) nutrient solution obtained in step 5) is centrifuged and filtered, it is thick that fusion protein recombined human hyaluronidase is made Product；
2) employment source protein specificity purification process purifies fusion protein recombined human hyaluronidase crude product.Purifying After, purity is more than 95%, and yield is more than 32%, and purification yield meets the requirement further done and medicine is subcutaneously injected.
Further, the recombined human hyaluronidase preparation method homologous with human body, centrifuge the rotating speed that uses for 5000-8000rpm, centrifugation time 6-10min.
Further, the recombined human hyaluronidase preparation method homologous with human body, takes the supernatant after centrifugation Filtered with three layers of filter membrane, filter sizes are respectively 0.8 μm, 0.45 μm, 0.22 μm.
The advantage of preparation method
1. the recombined human hyaluronic acid that the recombined human hyaluronidase digestion of the fragment of source protein containing someone obtains has human body Homology digestion end, can specifically bind with specific acceptor in human body and associated proteins, cause Human biology effect；
2. after people's source protein segment composition people's hyaluronidase, extend recombined human hyaluronidase partly declining in the reaction Phase；
3. compared with people's hyaluronidase rHuPH20 of polyethylene glycol chemistry coupling, fusion protein recombined human hyaluronic acid Enzyme is that use is safer entirely with people's source protein matter, although fusion protein recombined human hyaluronidase external activity (specific activity) drops It is low, but its activity in vivo is more superior；
4. fusion protein recombined human hyaluronidase after mammalian cell production, it can be achieved that commercial production level, The specific method of Purification of Human source protein fragment can be used to carry out highly purified, raising yield at the same time；
5. the fusion protein recombined human hyaluronidase can be used to digestion macromolecular hyaluronic acid, industrialized production recombined human Hyaluronic acid, can also be used to provide non-functional site, such as chemical coupling diagnosticum, medicine, treatment toxin, gene enter Human tissue organ.
(3) it is a kind of as follows with the homologous recombined human hyaluronic acid of human body, the recombined human hyaluronic acid structural formula：
Wherein n=3-10, structural formula both ends are and human body homology digestion end.
(4) a kind of side using the above-mentioned recombined human hyaluronidase Prepare restructuring people hyaluronic acid homologous with human body Method
Using recombined human hyaluronidase certain digestion technique and under the conditions of to macromolecular hyaluronic acid carry out digestion, Homology digestion end is obtained, and digestion technique is optimized and regulated and controled, obtains largely including 3-10 dissacharide units, molecule The hyaluronan oligosaccharides in 1000-4000D are measured, these specific small molecule amounts include the transparent of homology digestion end Matter acid fragment is recombined human hyaluronic acid.This method comprises the following steps：
1) purified water and NaCl are added into container, the concentration for adjusting NaCl in solution is 0.2-1.0mol/L, and pH is 5.0-7.0；
2) macromolecular hyaluronic acid dry powder is added into container, so that macromolecular hyaluronic acid concentration is 10- after dissolving 50g/L；
3) after macromolecular hyaluronic acid is completely dissolved, added into container homologous with human body as claimed in claim 1 Recombined human hyaluronidase, control concentration of enzymatic activity in 600-1000U/L；
4) container is placed in digestion in constant-temperature table after stirring evenly, digestion temperature is 35-40 DEG C, and the digestion time is 3- 4h, the pH of solution is 5.0-5.6 in control container during digestion；
5) it will digest solution after waiting digestion and carry out heating treatment, sterilization treatment is carried out to enzymolysis solution；
6) 95% ethanol solution precipitation, rear abandoning supernatant to be precipitated, with 95% are added into the solution in step 5) again Filter after ethanol dehydration, be dried in vacuo after suction filtration, obtained enzymolysis product is recombined human hyaluronidase.
Preferably, the method for Prepare restructuring people's hyaluronic acid, the macromolecular hyaluronic acid dry powder used in step 2) For purity selected from Fu Ruida productions in 90.0%-99.9%, molecular weight is the macromolecular hyaluronic acid of 200-2000KD.
Preferably, the method for Prepare restructuring people's hyaluronic acid, step 5) sterilising temp are 80 DEG C, and sterilization time is 30min。
Preferably, the method for Prepare restructuring people's hyaluronic acid, step 6) drying temperature are 60 DEG C
As a result：Macromolecular sodium hyaluronate solution is gel liquid after dissolving, molten in sample bottle after digestion for a period of time Liquid start it is thinning, present it is watery；Shown with HPLC testing results：Digestion products and sample enzyme class, sample concentration of enzymatic activity and Digestion process conditions have direct relation；The hyaluronic acid for including homology digestion end of the specific small molecule amount only obtained Fragment is only recombined human hyaluronic acid, just with human body activity.
The beneficial effects of the present invention are：
Recombined human hyaluronic acid prepared by this method due to self structure and divides in addition to having the function of normal transparent matter acid The specificity of son amount can also promote the secretion of human defensive's element 2 and antibacterial peptide to reach efficient suppression after human skin is quickly penetrated into System kills bacterium and the fungies such as Candida albicans, staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, so as to reduce by it Caused skin and mucosa infection and inflammatory reaction generation；Recombined human hyaluronic acid can be with after entering human skin at the same time As signaling molecule by cell membrane or intracellular specific bio-active molecule identifies, the signal of carrying correctly Amplify and be delivered to cell interior, and then cause Human biology effect, as recombined human hyaluronic acid can be with the TLR4 of human body Acceptor is combined, and reduces factor IL-1 β, IL-6 and the TNF-α secretion for inducing inflammation, and mutually tie with the CD44 acceptors of human body Close, control induces the cell migration and aggregation of inflammation, additionally it is possible to promote the generation of human peripheral macrophage IL-10, remove body The interior factor content for inducing inflammation, so as to suppress inflammatory reaction and slow down inflammatory stimulus effect, reaches enhancing skin and mucosa The effect of immunity.Since recombined human hyaluronic acid also has human skin the biological effect of obvious antibacterial anti-inflammatory, It can be widely used in the beauty and conditioning of the face in human body, oral cavity, hair, body and privates etc., make peace Overall coefficient higher, stability are more longlasting, the stronger daily chemical product of practicality.
Brief description of the drawings
Fig. 1 is recombined human hyaluronic acid structural formula figure.
Embodiment
In order to describe the technical content, the structural feature, the achieved object and the effect of this invention in detail, below in conjunction with embodiment It is explained in detail.
Embodiment 1
1. taking the triangular flask of some 250ml, 100ml purified waters are separately added into, marks and is added in backward each triangular flask NaCl, it is 0.5mol/L to make its concentration, and it is 7.0 to adjust pH；
2. respectively adding 3g macromolecular hyaluronic acid dry powder into every triangular flask, it is 30g/L to make its concentration, and dissolving is overnight；
3. after macromolecular hyaluronic acid is completely dissolved, into each triangular flask, recombined human hyaluronic acid enzyme liquid, control are added Concentration of enzymatic activity is in 400U/L；
It is placed on 4. 1500r/min is stirred evenly in constant-temperature table, 40 DEG C of digestion 4h, it is 5.0-5.6 during which to control pH；
5. enzymolysis solution temperature is increased to 80 DEG C after waiting digestion, 30min carries out enzyme-deactivating；
6. 95% ethanol solutions of 300ml precipitation is added into each sample again, rear abandoning supernatant to be precipitated, with 95% second Filtered after dehydration of alcohols, 60 DEG C of vacuum drying, obtain enzymolysis product, be detected with HPLC after suction filtration.
As a result：Macromolecular sodium hyaluronate solution is gel liquid after dissolving, and solution starts in sample bottle after digestion 3h It is thinning, present watery；HPLC testing results are shown：3-10 dissacharide units, molecular weight are practically free of in the saturating of 1000-4000D Bright matter acid oligose fragment.
Embodiment 2
1. taking the triangular flask of some 250ml, 100ml purified waters are separately added into, marks and is added in backward each triangular flask NaCl, it is 0.5mol/L to make its concentration, and it is 7.0 to adjust pH；
2. respectively adding 3g macromolecular hyaluronic acid dry powder into every triangular flask, it is 30g/L to make its concentration, and dissolving is overnight；
3. after macromolecular hyaluronic acid is completely dissolved, into each triangular flask, recombined human hyaluronic acid enzyme liquid, control are added Concentration of enzymatic activity is in 600U/L；
It is placed on 4. 1500r/min is stirred evenly in constant-temperature table, 40 DEG C of digestion 4h, it is 5.0-5.6 during which to control pH；
5. enzymolysis solution temperature is increased to 80 DEG C after waiting digestion, 30min carries out enzyme-deactivating；
6. 95% ethanol solutions of 300ml precipitation is added into each sample again, rear abandoning supernatant to be precipitated, with 95% second Filtered after dehydration of alcohols, 60 DEG C of vacuum drying, obtain enzymolysis product, be detected with HPLC after suction filtration.
As a result：Macromolecular sodium hyaluronate solution is gel liquid after dissolving, and solution starts in sample bottle after digestion 2h It is thinning, present watery；HPLC testing results are shown：Containing a large amount of 3-10 dissacharide units, molecular weight in the transparent of 1000-4000D Matter acid oligose fragment.
Embodiment 3
1. taking the triangular flask of some 250ml, 100ml purified waters are separately added into, marks and is added in backward each triangular flask NaCl, it is 0.5mol/L to make its concentration, and it is 7.0 to adjust pH；
2. respectively adding 3g macromolecular hyaluronic acid dry powder into every triangular flask, it is 30g/L to make its concentration, and dissolving is overnight；
3. after macromolecular hyaluronic acid is completely dissolved, into each triangular flask, recombined human hyaluronic acid enzyme liquid, control are added Concentration of enzymatic activity is in 1000U/L；
It is placed on 4. 1500r/min is stirred evenly in constant-temperature table, 40 DEG C of digestion 4h, it is 5.0-5.6 during which to control pH；
5. enzymolysis solution temperature is increased to 80 DEG C after waiting digestion, 30min carries out enzyme-deactivating；
6. 95% ethanol solutions of 300ml precipitation is added into each sample again, rear abandoning supernatant to be precipitated, with 95% second Filtered after dehydration of alcohols, 60 DEG C of vacuum drying, obtain enzymolysis product, be detected with HPLC after suction filtration.
As a result：Macromolecular sodium hyaluronate solution is gel liquid after dissolving, and solution is opened in sample bottle after digestion 1.8h Begin thinning, present watery；HPLC testing results are shown：Containing a large amount of 3-10 dissacharide units, molecular weight in the saturating of 1000-4000D Bright matter acid oligose fragment.
Embodiment 4
1. taking the triangular flask of some 250ml, 100ml purified waters are separately added into, marks and is added in backward each triangular flask NaCl, it is 0.5mol/L to make its concentration, and it is 7.0 to adjust pH；
2. respectively adding 3g macromolecular hyaluronic acid dry powder into every triangular flask, it is 30g/L to make its concentration, and dissolving is overnight；
3. after macromolecular hyaluronic acid is completely dissolved, into each triangular flask, recombined human hyaluronic acid enzyme liquid, control are added Concentration of enzymatic activity is in 1200U/L；
It is placed on 4. 1500r/min is stirred evenly in constant-temperature table, 40 DEG C of digestion 4h, it is 5.0-5.6 during which to control pH；
5. enzymolysis solution temperature is increased to 80 DEG C after waiting digestion, 30min carries out enzyme-deactivating；
6. 95% ethanol solutions of 300ml precipitation is added into each sample again, rear abandoning supernatant to be precipitated, with 95% second Filtered after dehydration of alcohols, 60 DEG C of vacuum drying, obtain enzymolysis product, be detected with HPLC after suction filtration.
As a result：Macromolecular sodium hyaluronate solution is gel liquid after dissolving, and solution is opened in sample bottle after digestion 1.5h Begin thinning, present watery；HPLC testing results are shown：3-10 dissacharide units, molecular weight are practically free of 1000-4000D's Hyaluronan oligosaccharides.
Embodiment 5
1. taking the triangular flask of some 250ml, 100ml purified waters are separately added into, marks and is added in backward each triangular flask NaCl, it is 0.5mol/L to make its concentration, and it is 7.0 to adjust pH；
2. respectively adding 3g macromolecular hyaluronic acid dry powder into every triangular flask, it is 30g/L to make its concentration, and dissolving is overnight；
3. after macromolecular hyaluronic acid is completely dissolved, into each triangular flask, recombined human hyaluronic acid enzyme liquid, control are added Concentration of enzymatic activity is in 1000U/L；
It is placed on 4. 1500r/min is stirred evenly in constant-temperature table, 30 DEG C of digestion 5h, it is 5.0-5.6 during which to control pH；
5. enzymolysis solution temperature is increased to 80 DEG C after waiting digestion, 30min carries out enzyme-deactivating；
6. 95% ethanol solutions of 300ml precipitation is added into each sample again, rear abandoning supernatant to be precipitated, with 95% second Filtered after dehydration of alcohols, 60 DEG C of vacuum drying, obtain enzymolysis product, be detected with HPLC after suction filtration.
As a result：Macromolecular sodium hyaluronate solution is gel liquid after dissolving, and solution is opened in sample bottle after digestion 1.6h Begin thinning, present watery；HPLC testing results are shown：3-10 dissacharide units, molecular weight are practically free of 1000-4000D's Hyaluronan oligosaccharides.
Embodiment 6
1. taking the triangular flask of some 250ml, 100ml purified waters are separately added into, marks and is added in backward each triangular flask NaCl, it is 0.5mol/L to make its concentration, and it is 7.0 to adjust pH；
2. respectively adding 3g macromolecular hyaluronic acid dry powder into every triangular flask, it is 30g/L to make its concentration, and dissolving is overnight；
3. after macromolecular hyaluronic acid is completely dissolved, into each triangular flask, recombined human hyaluronic acid enzyme liquid, control are added Concentration of enzymatic activity is in 1000U/L；
It is placed on 4. 1500r/min is stirred evenly in constant-temperature table, 50 DEG C of digestion 2.5h, it is 5.0-5.6 during which to control pH；
5. enzymolysis solution temperature is increased to 80 DEG C after waiting digestion, 30min carries out enzyme-deactivating；
6. 95% ethanol solutions of 300ml precipitation is added into each sample again, rear abandoning supernatant to be precipitated, with 95% second Filtered after dehydration of alcohols, 60 DEG C of vacuum drying, obtain enzymolysis product, be detected with HPLC after suction filtration.
As a result：Macromolecular sodium hyaluronate solution is gel liquid after dissolving, and solution is opened in sample bottle after digestion 2.2h Begin thinning, present watery；HPLC testing results are shown：3-10 dissacharide units, molecular weight are practically free of 1000-4000D's Hyaluronan oligosaccharides.
Embodiment 7
1. taking the triangular flask of some 250ml, 100ml purified waters are separately added into, marks and is added in backward each triangular flask NaCl, it is 0.5mol/L to make its concentration, and it is 7.0 to adjust pH；
2. respectively adding 3g macromolecular hyaluronic acid dry powder into every triangular flask, it is 30g/L to make its concentration, and dissolving is overnight；
3. after macromolecular hyaluronic acid is completely dissolved, into each triangular flask, normal transparent matter acid enzyme liquid is added, controls enzyme Active concentration is in 600U/L；
It is placed on 4. 1500r/min is stirred evenly in constant-temperature table, 40 DEG C of digestion 4h, it is 5.0-5.6 during which to control pH；
5. enzymolysis solution temperature is increased to 80 DEG C after waiting digestion, 30min carries out enzyme-deactivating；
6. 95% ethanol solutions of 300ml precipitation is added into each sample again, rear abandoning supernatant to be precipitated, with 95% second Filtered after dehydration of alcohols, 60 DEG C of vacuum drying, obtain enzymolysis product, be detected with HPLC after suction filtration.
As a result：Macromolecular sodium hyaluronate solution is gel liquid after dissolving, and solution is opened in sample bottle after digestion 2.5h Begin thinning, present watery；HPLC testing results are shown：3-10 dissacharide units, molecular weight are practically free of 1000-4000D's Hyaluronan oligosaccharides.
Embodiment 8
1. taking the triangular flask of some 250ml, 100ml purified waters are separately added into, marks and is added in backward each triangular flask NaCl, it is 0.5mol/L to make its concentration, and it is 7.0 to adjust pH；
2. respectively adding 3g macromolecular hyaluronic acid dry powder into every triangular flask, it is 30g/L to make its concentration, and dissolving is overnight；
3. after macromolecular hyaluronic acid is completely dissolved, into each triangular flask, normal transparent matter acid enzyme liquid is added, controls enzyme Active concentration is in 1000U/L；
It is placed on 4. 1500r/min is stirred evenly in constant-temperature table, 40 DEG C of digestion 4h, it is 5.0-5.6 during which to control pH；
5. enzymolysis solution temperature is increased to 80 DEG C after waiting digestion, 30min carries out enzyme-deactivating；
6. 95% ethanol solutions of 300ml precipitation is added into each sample again, rear abandoning supernatant to be precipitated, with 95% second Filtered after dehydration of alcohols, 60 DEG C of vacuum drying, obtain enzymolysis product, be detected with HPLC after suction filtration.
As a result：Macromolecular sodium hyaluronate solution is gel liquid after dissolving, and solution is opened in sample bottle after digestion 2.2h Begin thinning, present watery；HPLC testing results are shown：3-10 dissacharide units, molecular weight are practically free of 1000-4000D's Hyaluronan oligosaccharides.
The human body of 1 recombined human hyaluronic acid of test example active (inhibiting bacteria and diminishing inflammation) experiment
Purpose：Secreted, reduced using HT-29 epithelial cell lines measure given the test agent induction human defensive element 2 and antibacterial peptide Induce factor IL-1 β, IL-6 and the TNF-α secretion of inflammation, the bioactivity for promoting human peripheral macrophage IL-10 to produce. Method：The HT-29 epithelial cell lines (sample acceptor TLR4 and CD44 are positive) of human intestine's tumour are used in 37 DEG C of incubators Cultivated in RPMI nutrient solutions, supplement 10% calf serum (FBS).RPMI nutrient solutions contain given the test agent, for stimulating HT-29 Epithelial cell line production suppresses the factor of inflammatory reaction, and the specific factor uses enzyme linked immunological kit (Phoenix Pharmaceuticals (sample number=3)) are quantitative determined.Negative control is Normal Saline, and positive control is endotoxin LPS (10 mcg/ml), as shown in table 1.
The result of table 1HT-29 epithelial cell lines measure given the test agent is as follows：
Defensin 2 Antibacterial peptide L-1β IL-6 TNF-α IL-10
Nutrient solution 0.0±0.0 0.0±0.0 0.0 0.0 0.0 0.0
Embodiment one 2.0±0.2 2.0±0.3 2.0 150.0 12.0 1.5
Embodiment two 7.0±0.4 7.0±0.2 13.0 1000.0 95.0 5.0
Embodiment three 8.0±0.3 7.0±0.4 15.0 1200.0 100.0 5.5
Example IV 2.0±0.2 1.0±0.6 2.0 120.0 10.0 1.2
Embodiment five 1.0±0.2 2.0±0.2 2.5 200.0 10.0 1.0
Embodiment six 1.0±0.6 1.0±0.4 1.5 100.0 3.0 1.1
Embodiment seven 0.0±0.0 0.0±0.0 0.8 0.0 0.0 0.6
Embodiment eight 0.0±0.0 0.0±0.0 1.2 0.0 0.0 0.5
Negative control 0.0±0.0 0.0±0.0 1.0 0.0 0.0 0.8
Positive control 8.0±0.4 8.0±0.4 18.0 1500.0 150.0 6.0
Conclusion：Recombined human hyaluronic acid of the present invention ties up to the measure knot cultivated in nutrient solution through human body HT-29 epithelial cells Fruit shows：Only obtained using recombined human hyaluronidase digestion macromolecular hyaluronic acid under the conditions of certain digestion process Hyaluronic acid fragments of the small-molecular-weight with human body homology just have human body active, such as embodiment one and two；Present invention weight Group people's hyaluronic acid is acted on human skin, can promote the secretion of human defensive's element 2 and antibacterial peptide after absorption, in HT- Human defensive's element 2 and when small antibacterial peptide 24 interior highest increase by 8 times or so in the nutrient solution of 29 epithelial cell lines, can efficiently suppress Or bacterium and the fungies such as Candida albicans, staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa are killed, so as to reduce by them Caused skin and mucosa infection and the generation of inflammatory reaction；Recombined human hyaluronic acid is also used as after entering human skin at the same time Signaling molecule by cell membrane or intracellular specific bio-active molecule identify, the signal of carrying correctly is amplified And be delivered to cell interior, and then cause Human biology effect, can such as reduce factor IL-1 β, the IL-6 that induce inflammation and TNF-α secretion, can also control the cell migration and aggregation for inducing inflammation, additionally it is possible to promote human peripheral macrophage IL-10 Generation, remove in vivo induce inflammation factor content, so as to suppress inflammatory reaction and slow down inflammatory stimulus act on, reach Strengthen the effect of skin and mucosa immunity.
The bacteriostatic experiment of 2 recombined human hyaluronic acid of test example
Take Clinical isolation Escherichia coli, staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans a little, connect respectively Kind cultivates 18h in broth bouillon at 37 DEG C.(calf serum that 20% is added in Pseudomonas aeruginosa broth bouillon) takes Each bacterial strain nutrient broth culture of 18h cultures, is made into bacteria suspension and is used to test.Take sterilizing test tubes 11 respectively, the 1st Nutrient broth fluid nutrient medium 9ml is added, 2-10 branch adds 5ml, and the 11st adds 10ml, and separately sampled product solution 1ml adds Enter the 1st test tube, take 5ml to add the 2nd after mixing, be diluted to the 10th successively, the 11st is not added with sample as control. Often pipe adds escherichia coli suspension 0.1ml, and 24h is cultivated at a temperature of 37 DEG C, takes out observation bacterial growth situation.Golden yellow grape Coccus, Pseudomonas aeruginosa, Candida albicans are the same as above-mentioned Germicidal efficacy growing state.As test tube becomes cloudy, that is, represent bacterial growth, sample Product are without bacteriostasis；As test tube is limpid, expression bacterial growth is suppressed.Experimental result is as shown in table 2, the method for the present invention system Standby recombined human hyaluronic acid is equal to Clinical isolation Escherichia coli, staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans There is stronger bacteriostasis.
2 each sample solution of table corresponds to last limpid test tube numbering in bacteriostatic test：
Escherichia coli Staphylococcus aureus Pseudomonas aeruginosa Candida albicans
Embodiment one - - - -
Embodiment two 7 6 6 7
Embodiment three 7 6 6 7
Example IV 1 - - 1
Embodiment five 1 - - 1
Embodiment six - - - 1
Embodiment seven - - - -
Embodiment eight - - - -
Control group - - - -
Note："-" represents not limpid.
By above-mentioned limpid broth tubes and control group the transferred species broth agar plates after diluting according to a certain percentage, observation 24h, the Cmin of no bacterial growth is bacteriocidal concentration, is denoted as C, unit mg/mL, the results are shown in Table 3, illustrates this hair Bright recombined human hyaluronic acid to be clinically separated common Bacteria skin infection strain Escherichia coli, staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans has stronger bactericidal effect, so as to reduce the skin and mucosa infection caused by them and the hair of inflammatory reaction It is raw, play prevention and the effect of conditioning skin discomfort.
3 each sample solution sterilization conditions of table：
C (Escherichia coli) C (staphylococcus aureus) C (Pseudomonas aeruginosa) C (Candida albicans)
Embodiment one - - - -
Embodiment two 0.11 0.12 0.22 0.08
Embodiment three 0.13 0.11 0.20 0.12
Example IV 0.83 - - 0.90
Embodiment five 0.87 - - 0.95
Embodiment six - - - 1.04
Embodiment seven - - - -
Embodiment eight - - - -
Control group - - - -
Note："-" indicates no bactericidal properties.
The foregoing is merely the embodiment of the present invention, not thereby limits scope of patent protection of the invention, every utilization The equivalent structure or equivalent flow shift that present specification is made, is directly or indirectly used in other relevant technologies Field, is included within the scope of the present invention.

Claims (10)

1. a kind of and homologous recombined human hyaluronic acid of human body, it is characterised in that the recombined human hyaluronic acid structural formula is as follows：
Wherein n=3-10, structural formula both ends are and human body homology digestion end.
2. the recombined human hyaluronidase Prepare restructuring people hyaluronic acid homologous with human body is used as claimed in claim 1, its It is characterized in that, the extracellular parts of the recombined human hyaluronidase behaviour hyaluronidase PH20 that the present invention uses and people's source protein The fusion protein of (human albumin or immunoglobulin) fragment, its amino acid sequence contain 644 amino acid residues, have human body The advantages of homology is high, long half time.
3. the recombined human hyaluronidase homologous with human body as claimed in claim 2, it is characterised in that preparation method is included such as Lower step：
1) target gene of fusion protein recombined human hyaluronidase is building up to by PCR method by the zooblast table rich in GC Up in carrier pMH3, pMH4 or pMH5；
2) after preparing expression plasmid, it is stably transfected into the Chinese hamster ovary celI of serum free suspension culture；Screened by G418, use pipette tips Manual picking stable clone is into 96 orifice plates；
3) when cell confluency degree is more than 50%, serum-free fresh culture is replaced；
4) after when 3-6 is small, media samples are collected, expression quantity is detected by antibody dot blot or ELISA method, selects expression quantity Highest multiple clones, continue to carry out free serum culture in the small shaking flask in sharp bottom and do mitotic stability research；
5) select the stable clone of passage and carry out amplification cultivation in 5-8L rip current type reactors, selected in each batch has a good harvest liquid The nutrient solution for taking hyaluronidase activity concentration to reach 600-1000U/L.
4. the recombined human hyaluronidase preparation method homologous with human body as claimed in claim 3, it is characterised in that in step 5) following steps are further included after：
1) nutrient solution obtained in step 5) is centrifuged and filtered, fusion protein recombined human hyaluronidase crude product is made；
2) employment source protein specificity purification process purifies fusion protein recombined human hyaluronidase crude product.
5. the recombined human hyaluronidase preparation method homologous with human body as claimed in claim 3, it is characterised in that centrifugation is adopted Rotating speed is 5000-8000rpm, centrifugation time 6-10min.
6. the recombined human hyaluronidase preparation method homologous with human body as claimed in claim 3, it is characterised in that take centrifugation Supernatant afterwards is filtered with three layers of filter membrane, and filter sizes are respectively 0.8 μm, 0.45 μm, 0.22 μm.
7. a kind of prepared such as claim 1 institute using recombined human hyaluronidase homologous with human body as claimed in claim 2 The method for stating recombined human hyaluronic acid, it is characterised in that this method comprises the following steps：
1) purified water and NaCl are added into container, the concentration for adjusting NaCl in solution is 0.2-1.0mol/L, pH 5.0- 7.0；
2) macromolecular hyaluronic acid dry powder is added into container, so that macromolecular hyaluronic acid concentration is 10-50g/L after dissolving；
3) after macromolecular hyaluronic acid is completely dissolved, weight homologous with human body as claimed in claim 1 is added into container Group people's hyaluronidase, control concentration of enzymatic activity is in 600-1000U/L；
4) container is placed in digestion in constant-temperature table after stirring evenly, digestion temperature is 35-40 DEG C, and the digestion time is 3-4h, enzyme The pH of solution is 5.0-5.6 in control container during cutting；
5) it will digest solution after waiting digestion and carry out heating treatment, sterilization treatment is carried out to enzymolysis solution；
6) 95% ethanol solution precipitation, rear abandoning supernatant to be precipitated, with 95% ethanol are added into the solution in step 5) again Filter after dehydration, be dried in vacuo after suction filtration, obtained enzymolysis product is recombined human hyaluronidase.
8. the method for Prepare restructuring people hyaluronic acid as claimed in claim 7, it is characterised in that the macromolecular used in step 2) Hyaluronic acid dry powder is selected from the purity of Fu Ruida productions in 90.0%-99.9%, and molecular weight is that the macromolecular of 200-2000KD is saturating Bright matter acid.
9. the method for Prepare restructuring people hyaluronic acid as claimed in claim 7, it is characterised in that step 5) sterilising temp is 80 DEG C, sterilization time 30min.
10. the method for Prepare restructuring people hyaluronic acid as claimed in claim 7, it is characterised in that step 6) drying temperature is 60℃。
CN201711144684.2A 2017-11-17 2017-11-17 A kind of recombined human hyaluronic acid and its production method Pending CN107988183A (en)

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