CN104342420A - Recombinant long-acting human hyaluronidase, and encoding gene, production method and application thereof - Google Patents
Recombinant long-acting human hyaluronidase, and encoding gene, production method and application thereof Download PDFInfo
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- CN104342420A CN104342420A CN201310325056.XA CN201310325056A CN104342420A CN 104342420 A CN104342420 A CN 104342420A CN 201310325056 A CN201310325056 A CN 201310325056A CN 104342420 A CN104342420 A CN 104342420A
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- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a recombinant long-acting human hyaluronidase. The recombinant long-acting human hyaluronidase is a fusion protein PH20-HSA or PH20-IgFc of a human hyaluronidase PH20 extracellular part and a human protein (albumin HSA fragment or immunoglobulin IgG2Fc fragment), and the amino acid sequences of the PH20-HSA and the PH20-IgFc are represented by SEQ ID No. 4 and SEQ ID No. 5 respectively, and have the advantages of high in-vivo safety and long half-life. The invention also discloses an encoding gene, an expression vector, a host cell, a production method and a stable preparation of the recombinant long-acting human hyaluronidase. The achievement of industrial level and the obtaining of purified products with active energy are realized through expressing by using GC-rich high expression system and through producing Chinese hamster cells (CHO). The invention further discloses an application of the recombinant long-acting human hyaluronidase in cooperative dosage and the production of micro-molecular hyaluronic acid.
Description
Technical field
The present invention relates generally to biological technical field, particularly relates to a kind of recombinant long-acting people Unidasa, its encoding gene, production method and application.
Background technology
Hyaluronic acid is mucopolysaccharide form main in corium, the linear polysaccharide be made up of the disaccharide unit D-Glucose aldehydic acid repeated and N-acetyl-glucosamine.Hyaluronic acid can remain above the moisture of own wt 500 times, forms the solution of very thickness, is filled within extracellular matrix collagen fabric skeleton, affects the mass transfer characteristics of extracellular matrix.Extracellular matrix is that many medicines carry out hypodermic major obstacle.Hyaluronic viscogel is rich in the solid-state pedestal that collagen fabric is formed and embedding, cause certain resistance to mass transfer, limit pharmacokinetics and hypodermic liquid volume can be carried out from same site, and arriving speed and the quantity of blood vessel.
Unidasa is the hyaluronic enzyme family of degraded, and by the hyaluronic hydrolysis of catalysis, Unidasa reduces hyaluronic viscosity, reversibly changes the structure of corium, improves the permeability of tissue thus.Hyaluronic acid half life of enzyme 15-20h, update time is short in vivo, and skin can be repaired fast.Extracellular matrix barrier administration of getting through based on Unidasa is a kind of new and effective administration platform.Different according to the mechanism of action and degraded product, Unidasa can be divided three classes, the first kind comprises β-D-acetyl-D-hexosaminidase, becomes tetrose as main end product high molecular degradation of substrates.Testicular hyaluronidase is the prototype of this kind, and it can catalysis transglycosylation, therefore, also can form hexose, disaccharides and eight carbon sugar in the hyaluronic process of hydrolysis.Equations of The Second Kind, the Unidasa being representative with leech Unidasa.3rd class, bacterial hyaluronidase is a kind of lyase.
Since 1940s, the spreading factor containing hyaluronidase activity extracted in animal tissues is as urging diffusant for clinical administration, but its range of application is defined in emergency situation always, mainly because the misgivings to other impurity components in the not exclusively clear and definite zymin of composition.Animal tissues extracts effective constituent in hyaluronidase preparation and is less than 1%, and the overwhelming majority is gous impurities albumen.After gender bender's Unidasa gene discovery, the recombination human source Unidasa (rHuPH20) in DNA source is adopted to replace the Unidasa of animal source purifying, reduce immunogenicity, improve security, range of application also expands thereupon, is mainly used in hypodermoclysis, periocular surgery anesthesia, chemical drug and biological medicament are urged to ooze, the absorption of contrast medium in urography.
Hyaluronic enrichment is had at many solid tumors (comprising prostate cancer, mammary cancer, carcinoma of the pancreas, colorectal carcinoma and nonsmall-cell lung cancer) cell surface, be rich in hyaluronic cell micro-environment and often there is with between tumor development association, provide the condition of malignant growth diffusion.Adopt the hyaluronic acid integument of the tissue of hyaluronic acid ferment treatment tumor growth or organ or process tumor cell surface and combine anti-tumor medicine, providing a kind of anticancer New Policy.
But the transformation period is shorter than 1 minute in the rHuPH20 body inner blood do not transformed, cannot input through blood and realize the effect of interior therapeutic, existing people uses polyoxyethylene glycol chemistry coupling rHuPH20, produces long-acting people's Unidasa, and treats tumour for human clinical trial.
On the other hand, Unidasa can be degraded the artificial restructuring macromolecule hyaluronic acid produced, and produces small-molecular-weight hyaluronic acid.Small-molecular-weight hyaluronic acid, the small-molecular-weight hyaluronic acid oligosaccharides (oHA) comprised containing 3-10 dissacharide units can activate endothelial cell surface CD44 and promote endothelial cell proliferation, can also work in coordination with mutually with vascular endothelial growth factor (VEGF), promoting endotheliocyte angiogenesis, is the form of hyaluronic acid having biological effect.
Therefore, small-molecular-weight hyaluronic acid is widely used at cosmetic field, because its molecular weight is little, can infiltrate dermal layer of the skin corium, have stronger biological effect.And industrial production small-molecular-weight hyaluronic acid many employings soda acid degraded at present or ultrasonic degradation.
Summary of the invention
The object of this invention is to provide that security in a kind of body is high, the recombinant long-acting people Unidasa of long half time.
For achieving the above object, the present invention adopts following technical scheme: a kind of recombinant long-acting people Unidasa, the fusion rotein of behaviour Unidasa PH20 extracellular part and people's source protein, described people's source protein is human albumin HSA fragment or human normal immunoglobulin IgG2Fc fragment; The fusion rotein of people's Unidasa PH20 extracellular part and human albumin HSA fragment is PH20-HSA, and its aminoacid sequence is as shown in SEQ ID No.4; The fusion rotein of people's Unidasa PH20 extracellular part and human normal immunoglobulin IgG2Fc fragment is PH20-IgFc, and its aminoacid sequence is as shown in SEQ ID No.5.
Second object of the present invention is to provide a kind of encoding gene of above-mentioned recombinant long-acting people Unidasa, adopts following technical scheme: its gene order is as shown in SEQ ID No.6 or SEQ ID No.7.
3rd object of the present invention is to provide a kind of expression vector and the host cell that comprise gene order shown in SEQ ID No.6 or SEQ ID No.7.
4th object of the present invention is to provide the production method of above-mentioned people's Unidasa, adopt following technical scheme: use animal cell expression vectors pMH3, pMH4 or pMH5 expressing gene sequence SEQ ID No.6 or SEQ ID No.7 in China ground hamster cell CHO being rich in GC, then carry out separation and purification and get final product; The gene order of described expression vector pMH3, pMH4 or pMH5 is respectively as shown in SEQ ID No.8,9,10.
Further, described PH20-HSA uses ProteinA affinity purification method to carry out separation and purification; Described PH20-IgFc adopts ion hydrophobic series connection blue glue series connection ion method of connecting to carry out separation and purification.
5th object of the present invention is to provide the preparation that a kind of stably stored has above-mentioned people's Unidasa, adopt following technical scheme: described preparation comprises PH20-HSA or PH20-IgFc of 150-1500IU, 10mM Na2HPO4,0.03%CaCl2,0.1%EDTA-Na2,145mM NaCl, 0.1%HSA, and pH value is 5.5-7.5.
6th object of the present invention is to provide a kind of compound preparation, adopts following technical side's scheme: compound preparation comprises preparation and the particular treatment medicine that above-mentioned stably stored has people's Unidasa.
7th object of the present invention is to provide above-mentioned recombinant long-acting people Unidasa and is coordinating the application in administration, adopts following technical side's scheme: recombinant long-acting people hyaluronidase activity is delivered to the tissue of human albumin or human normal immunoglobulin combination, organ, position when not being degraded; Or by particular treatment useful for drug delivery to the tissue at disease place, organ or position.
8th object of the present invention is to provide the application of above-mentioned recombinant long-acting Unidasa, IgFc and HSA fragment wherein provides that chemical coupling diagnostic reagent is carried out in NOT-function site, photographic developer, medicine, treatment toxin, gene enter diseased tissue organ.
9th object of the present invention is to provide the application of above-mentioned recombinant long-acting people Unidasa in industrial production micromolecule hyaluronic acid, adopts following technical scheme: described recombinant long-acting people Unidasa produces micromolecule hyaluronic acid as toolenzyme degraded macromole hyaluronic acid.
Owing to adopting technique scheme, the present invention at least has the following advantages:
(1), after human normal immunoglobulin IgG2Fc fragment and human albumin HSA segment composition people Unidasa, the transformation period in the blood extending people's Unidasa, disease location can be transported to through blood or additive method, extension of validity.
(2) human normal immunoglobulin IgG2Fc fragment and human albumin HSA fragment can combine with specific acceptor in human body and associated proteins, people's Unidasa of fusion can be carried to these positions.
(3) compared with people's Unidasa rHuPH20 of polyoxyethylene glycol chemistry coupling, people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc) and people's Unidasa human albumin fusion rotein (PH20-HSA) are total man's source protein matter, use safer in body.
(4) although compared with Unidasa rHuPH20, people's Unidasa human albumin fusion rotein (PH20-HSA) and people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc) external activity (specific activity) reduce, but their activity in vivo is more superior.
(5) after PH20-HSA and PH20-IgFc mammalian cell of the present invention is produced, the specific method purifying of purifying HSA and IgFc can be adopted, in production method and production prices, be better than the method that polyoxyethylene glycol chemistry coupling rHuPH20 produces long-acting people's Unidasa.
(6) can the long-acting fusion rotein of suitability for industrialized production people Unidasa be a technological challenge to Microbial Expression Systems, even if use mammalian cell liquid to be difficult to the product reaching industrialized level and obtain active energy purifying.The present invention adopt the animal cell expression vectors that is rich in GC and China's ground hamster cell (CHO) successful commercialization produce active can people's Unidasa human albumin fusion rotein (PH20-HSA) of purifying and people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc).
(7) described people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc) and people's Unidasa human albumin fusion rotein (PH20-HSA) can be used to do long-acting toolenzyme of not easily degrading, degraded macromole hyaluronic acid, suitability for industrialized production micromolecule hyaluronic acid.
(8) described people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc) and people's Unidasa human albumin fusion rotein (PH20-HSA) IgFc and HSA fragment can chemical coupling diagnostic reagent be carried out in NOT-function site, photographic developer, medicine, treatment toxin, gene enter diseased tissue organ with providing.
Accompanying drawing explanation
Above-mentioned is only the general introduction of technical solution of the present invention, and in order to better understand technique means of the present invention, below in conjunction with accompanying drawing and embodiment, the present invention is described in further detail.
Fig. 1 is fusion rotein PH20-IgFc37 of the present invention DEG C stability test result schematic diagram.
Fig. 2 is fusion rotein PH20-HSA37 of the present invention DEG C stability test result schematic diagram.
Fig. 3 is that the specific activity of fusion rotein PH20-IgFc of the present invention and contrast people Unidasa is in big white mouse experiment in vivo result schematic diagram.
Fig. 4 is the radioactivity absorption distribution situation schematic diagram of intravenous injection PH20-IgFc animal (A), intravenous injection PH20-HSA animal (B), intravenous injection PH20 animal (C) subcutaneous injection site absorption 99mTc-sestamibi.
Fig. 5 is people's Unidasa PH20 and the blood clearance situation of people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein PH20-IgFc in rat body of 99mTc mark.
Fig. 6 is people's Unidasa PH20 and the distribution situation of people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein PH20-IgFc in rat body of 99mTc mark.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described, are to be understood that preferred embodiment described herein is only for instruction and explanation of the present invention, is not intended to limit the present invention.
The present invention mainly utilizes is rich in GC animal cell expression technology (WO/2008/091276) and zooblast reactor (WO2006/138143 and WO2007/142664) technology, produce rapidly long-acting people's Unidasa of non-polyoxyethylene glycol chemistry coupling rHuPH20, comprise people's Unidasa human albumin fusion rotein PH20-HSA and people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein PH20-IgFc.People's Unidasa PH20, its extracellular partial amino-acid series is as shown in SEQ ID No.1; The aminoacid sequence of human albumin HSA fragment is as shown in SEQ ID No.2; The aminoacid sequence of human normal immunoglobulin IgG2Fc fragment is as shown in SEQ ID No.3.
Embodiment one
Object: gene constructed, the expression of fusion rotein PH20-HSA or PH20-IgFc, immunology detection, Activity determination and reactor are produced.
Method: by the method for PCR by structure gene PH20-HSA(SEQIDNo.6) or PH20-IgFc(SEQIDNo.7) be building up to be rich in GC animal cell expression vectors pMH3,4,5(SEQIDNo.8,9,10) in.After preparing this several expression plasmid in a small amount, be stably transfected in the Chinese hamster ovary celI of serum free suspension cultivation.Screened by G418, with the manual picking stable clone of rifle head in 96 orifice plates.When cell confluency degree is greater than 50%, change serum-free fresh culture; After 3-6 hour, collect media samples, detect expression amount by anti-igg 2Fc antibody dot blot or ELISA method, the clone selecting expression amount the highest is multiple, continues in the little shaking flask in the point end, to carry out serum-free culture and do mitotic stability research; Select stable being cloned in 5L rip current type reactor of going down to posterity to increase.
Result: (1) fusion rotein PH20-IgFc or PH20-HSA successful expression in Chinese hamster ovary celI, dot blot and western blot result show to screen fusion rotein PH20-IgFc or PH20-HSA to stablize the band molecular size range that high-expression clone produces correct; (2) high-expression clone carries out serum-free culture and goes down to posterity stable in the little shaking flask in the point end; (3) stable high-expression clone carries out feeding culture amplification in 5L rip current type reactor, and in each batch of good harvest liquid, hyaluronidase activity reaches 600-1000 activity unit/milliliter, reaches commercial production level.
Conclusion: use the animal cell expression vectors pMH3,4,5 being rich in GC, fusion rotein PH20-IgFc or PH20-HSA successful expression in Chinese hamster ovary celI, in each batch of good harvest liquid, hyaluronidase activity reaches 600-1000 activity unit/milliliter, can meet suitability for industrialized production.
Embodiment two
Object: the separation and purification of fusion rotein PH20-HSA or PH20-IgFc.
Method: the separation and purification of protein strategy that the present embodiment adopts is that (1) PH20-IgFc mainly adopts ProteinA affinity purification; (2) PH20-HSA adopts the purifying process of the hydrophobic blue glue of ion series connection.
(1) PH20-IgFc receives liquid purifying.
After results nutrient solution, adopt the centrifugal 6min of 5000rpm, get centrifuged supernatant and filter, three metafiltration membrane pore size are respectively 0.8 μm, 0.45 μm, 0.22 μm.Loading, to the Mabselect pillar having used balance liquid A liquid (20mM Tris+100mM NaCl, pH7.4) to balance, after drip washing is constant to baseline, adopts elutriant B liquid (100mM Gly+50mM Arg+0.01%Tween80, pH3.5) to carry out wash-out.The separation and purification situation of product is detected by SDS-PAGE.
(2) PH20-HSA receives liquid purifying
After PH20-HSA cell culture fluid receives liquid, adopt the centrifugal 6min of 5000rpm, get centrifuged supernatant and filter, three metafiltration membrane pore size are respectively 0.8 μm, 0.45 μm, 0.22 μm.Adopt Millipore30kDa film bag to be carried out by the supernatant after filtering concentratedly changing liquid, displacement liquid is anion column QSFF level pad.
By concentrated change liquid after protein liquid loading to using A liquid (20mMTris+50mMNaCl, pH7.4) the QSFF post balanced, after drip washing is constant to baseline, adopt C liquid (20mMTris+100mM NaCl, pH7.0) baseline is washed till in advance constant, with E liquid (20mM Tris+450mM NaCl, pH7.0) wash-out, collect elution peak.
QSFF elution peak is added (NH
4)
2sO
4to 0.5M, add CaCl
2to 0.1mM, adjustment pH to 7.0, with 2ml/min flow velocity loading to using A liquid (20mM Tris+0.5M (NH
4)
2sO
4+ 0.1mMCaCl
2, pH7.0) and the Phenyl post that balanced, after drip washing is constant to baseline, E liquid (20mM Tris+0.25M (NH
4)
2sO
4+ 0.1mMCaCl
2, pH7.0) and carry out prewashing, with B liquid (20mM Tris+0.1mMCaCl
2, pH7.0) and carry out wash-out.
It is 20mS/cm that Phenyl elution peak component is diluted with water to conductance, and adjusts pH to 7.0 with 1M HCl/1M NaOH.Loading is to using I liquid (10mM Tris+150mM KCl, pH7.0) Blue post is balanced, loading, with F liquid (10mM Tris+600mM KCl, pH8.0) carry out wash-out, adopt H liquid (10mM Tris+1.5M KCl+50% ethylene glycol, pH8.0) to carry out Strip, flow velocity is 1ml/min, collects elution peak.The separation and purification situation of product is detected by SDS-PAGE.
Result: adopt above-mentioned separation and purification strategy, the purity of fusion rotein PH20-IgFc or PH20-HSA is more than 95%, and yield is greater than 30%.
Conclusion: fusion rotein PH20-IgFc and PH20-HSA can adopt IgFc and HSA specificity purification process to carry out purifying, and purification yield meets the requirement doing subcutaneous injection medicine further.
Embodiment three
Object: the preparation of fusion rotein PH20-IgFc or PH20-HSA and In vitro and in vivo activity research
Method: the pharmaceutical formulation of the fusion rotein PH20-IgFc injection liquid of Unidasa is as follows:
PH20-IgFc+10mM Na2HPO4+0.03%CaCl2+0.1%EDTA-Na2+145mM NaCl+0.1%HSA，pH5.5-7.5
Being divided by solution after 500U/mlPH20-IgFc filtration is filled in 2ml ampere bottle, and 25 bottles of solution are placed in 37 DEG C, and humidity is in the accelerated test incubator of 75 ± 5%.Sampled according to 2010 editions pharmacopeia annex hyaluronidase Activity determination at the 0th, 7,14,21,28,35,42 day respectively, stability test result (table 1, Fig. 1) shows, and from the 28th day, the activity of Unidasa occurred that significance declines (about 15%).
Show 1PH20-IgFc37 DEG C of stability test result
The pharmaceutical formulation of the fusion rotein PH20-HSA injection liquid of Unidasa is as follows:
150-1500IU PH20-HSA+10mM Na2HPO4+0.03%CaCl2+0.1EDTA-Na2+145mM NaCl+0.1%HSA，pH5.5-7.5
Stability test: divided by the solution after 500U/mlPH20-HSA filtration and be filled in 2ml ampere bottle, 25 bottles of solution are placed in 37 DEG C, and humidity is in the accelerated test incubator of 75 ± 5%.Sampled according to 2010 editions pharmacopeia annex hyaluronidase Activity determination at the 0th, 7,14,21,28,35,42 day respectively, stability test result (table 2, Fig. 2) shows, and from the 28th day, the activity of Unidasa occurred that significance declines (about 15%).
Show 2PH20-HSA37 DEG C of stability test result
| Time | 1 | 2 | 3 | Mean value (U/ml) | Deviation | CV |
| 1 | 468 | 513 | 481 | 487.17 | 22.88 | 4.70 |
| 7 | 420 | 520 | 534 | 491.33 | 62.17 | 12.65 |
| 14 | 496 | 547 | 507 | 516.67 | 26.84 | 5.19 |
| 21 | 443 | 489 | 502 | 478.00 | 31.00 | 6.49 |
| 28 | 486 | 434 | 450 | 456.67 | 26.63 | 5.83 |
| 35 | 485 | 459 | 437 | 460.33 | 24.03 | 5.22 |
| 42 | 447 | 390 | 448 | 428.33 | 33.20 | 7.75 |
Specific activity and the contrast people Unidasa of fusion rotein PH20-IgFc and PH20-HSA have done external comparative study, by setting up trypan blue diffusion model in young mouse body, determine the activity in vivo of PH20-IgFc or PH20-HSA, also measure for the transformation period in blood plasma.
Result: external in vitro preparation stability experimental result shows, the activity of fusion rotein PH20-IgFc and the PH20-HSA stability of 37 degree in above preparation is all greater than 20 days, and loss of activity is less than 10%.
External in vitro experimental result also shows, people's hyaluronic acid specific enzyme activity that contrast is produced is 110000 units/milligram albumen, the specific activity of fusion rotein PH20-IgFc is 40000 units/milligram albumen, the specific activity of PH20-HSA is 20000 units/milligram albumen, and people's hyaluronic acid specific enzyme activity that the specific activity of fusion rotein PH20-IgFc is produced than contrast is low by about 40%; And PH20-HSA specific activity is only people's Unidasa about 10% that contrast is produced.
In big white mouse body, Ink vessel transfusing experimental result also shows, people's hyaluronic acid half life of enzyme that contrast is produced is very short, be only 1-2min, the transformation period of fusion rotein PH20-IgFc and PH20-HSA reaches 2.5h, and in the blood of fusion rotein PH20-IgFc and PH20-HSA, the transformation period is about 100 times that contrast the people's hyaluronic acid specific enzyme activity produced.
Conclusion: the activity of fusion rotein PH20-IgFc and the PH20-HSA stability of 37 degree in above preparation is greater than 20 days, the external specific activity of fusion rotein PH20-IgFc and PH20-HSA is lower than the specific activity contrasting the people's Unidasa produced, and the transformation period of fusion rotein PH20-IgFc and PH20-HSA is much larger than contrasting the people's hyaluronic acid half life of enzyme produced.
The present invention adopts hyaluronic acid degradation method that is active and Westernblot to determine the Half-life in vivo of fusion rotein.PH20-IgFc or PH20-HSA comparatively PH20 lifetime (i.e. transformation period) in Mice Body obviously extends, and its vivo degradation hyaluronic acid effect of prompting PH20-IgFc or PH20-HSA also should comparatively extend by PH20, and namely drug effect extends.PH20-IgFc or PH20-HSA is the degraded hyaluronic medicine more long-acting than PH20.
Embodiment four
Object: the N measuring fusion rotein PH20-IgFc and PH20-HSA holds 15 aminoacid sequences
Method: do not dye after sample separation with SDS-PAGE electrophoresis, goes to pvdf membrane with CAPS damping fluid by its electricity, and coomassie brilliant blue staining, rinsing, decolouring are to without blue background, for subsequent use after seasoning; Target protein band is cut, uses ABIPROCISETM494cLC instrument, carry out N-terminal sequencing according to N-terminal sequencing standard method (SCI-S-006).
Result: sequencing result display N terminal amino acid sequence is that Leu-Asn-Phe-Arg-Ala-Pro-Pro-Val-Ile-Pro-Asn-Val-Pro-Phe-Leu is consistent with theoretical value.
Embodiment five
Object: people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc) and people's Unidasa human albumin fusion rotein (PH20-HSA) macromole hyaluronic acid of degrading is active.
Method:
1) get the triangular flask of 3 250ml, add 100ml purified water respectively, be labeled as 1,2, No. 3, in each triangular flask, add 1.17gNaCl, make its concentration be 0.2mol/L, regulate pH to be 7.0.
2) add 1-3g hyaluronic acid dry powder to often propping up in triangular flask, make its concentration be 10-30g/L, dissolving is spent the night.
3) after hyaluronic acid dissolves completely, in 1,2, No. 3 bottle, add sample enzyme liquid, in sample, enzyme amount is respectively 6000U, 12000U, 18000U, and enzyme concn is respectively 60U/ml, 120U/ml, 180U/ml; Stir and be placed in shaking table, 40 DEG C of enzymes cut 4h.
4) wait for that solution temperature is increased to 80 DEG C after cutting end by enzyme, 30min carries out enzyme-deactivating;
5) in three samples, respectively add 300ml95% ethanolic soln precipitation, rear abandoning supernatant to be precipitated, with suction filtration after 95% ethanol dehydration.
6) 60 DEG C of vacuum-dryings after suction filtration, detect with HPLC.
Result: after dissolving, sodium hyaluronate solution is gel liquid, and when enzyme cuts 2h, in 3 triangular flasks, solution starts thinning, presents watery; HPLC detected result shows: hyaluronic molecular weight is relevant with the consumption of enzyme, and more macromolecule is less for the consumption of enzyme.(500KDa during 60U/ml; 300KDa during 120U/ml; 200KDa during 180U/ml)
Conclusion: described people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc) and people's Unidasa human albumin fusion rotein (PH20-HSA) can be used to do long-acting toolenzyme of not easily degrading, degraded macromole hyaluronic acid, suitability for industrialized production micromolecule hyaluronic acid.
Embodiment six
Object: the effect of application of radiation Trace imaging comparative studies biological infiltration accelerating agent people Unidasa, people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc), people's Unidasa human albumin fusion rotein (PH20-HSA) enhanced tissue drug absorption.
Method: people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc), people's Unidasa human albumin fusion rotein (PH20-HSA), people's Unidasa (PH20) injects four groups of healthy ICR mouse respectively through vein, treat latter 2 hours, at the tracer agent methoxy isobutyl isonitrile (99mTc-sestamibi) that left or right thigh position subcutaneous injection 99mTc marks, the special γ camera of application high resolution animalcule obtains live body mouse whole body imaging, after observing the subcutaneous injection of radiolabeled drug simulated compound, absorption rate and body tissue distribute.Note: 99mTc-sestamibi for many years for tumor drug resistance evaluation as medicine surrogate, applies this method and detects Unidasa strengthening drug absorption, have the advantages such as sensitive, quick, directly perceived, reliable.
Result: as shown in Figure 4, the radioactivity of intravenous injection PH20-IgFc animal (A), intravenous injection PH20-HSA animal (B), intravenous injection PH20 animal (C) subcutaneous injection site absorption 99mTc-sestamibi absorbs distribution situation display, in PH20-IgFc and PH20-HSA treatment group Mice Body, the increased radioactivity of 99mTc-sestamibi and major organs radioactivity level are apparently higher than PH20 animal, through image quantitative analysis, treat latter 2 hours, in promotion 99mTc-sestamibi subcutaneous absorption performance, PH20-IgFc and PH20-HSA comparatively PH20 is strong 2.3 times.
Conclusion: due to the change of PH20-IgFc and PH20-HSA blood halflife, PH20-IgFc and PH20-HSA, in promotion drug absorption, is obviously better than PH20.
Embodiment seven
Object: people's Unidasa (PH20) of research 99mTc mark and the plasma half-life of people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc).
Method: (PH20 and people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc) vein are injected in Sprague Dawley rat body the people's Unidasa marked by 99mTc, use living imaging instrument to measure their plasma half-life (Blood clearance (%peak) of99mTc-labeled PH20-Fc andPH20).
Result: as shown in Figure 5, the plasma half-life of people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc) of 99mTc mark is obviously long than the plasma half-life of people Unidasa (PH20).
Conclusion: the plasma half-life of people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc) is obviously long than the plasma half-life of people Unidasa (PH20).
Embodiment eight
Object: people's Unidasa (PH20) of research 999mTc mark and people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc) the absorption distribution in rat body.
Method: (PH20 and people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc) vein are injected in Sprague Dawley rat body the people's Unidasa marked by 99mTc, after 3 hours, they distribute in the absorption of Different Organs to use the quantitative assay of living imaging instrument.
Result: as shown in Figure 6, people's Unidasa (PH20) of 99mTc mark is different with the absorption distribution of people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc) in rat body.
The absorption distribution in rat body of people's Unidasa (PH20) that conclusion: 99mTc marks and people's Unidasa human normal immunoglobulin IgG2Fc fragment fusion protein (PH20-IgFc) is different.
People's Unidasa human albumin fragment (PH20-HSA) and Unidasa human normal immunoglobulin Fc fragment (PH20-IgFc) are merged in the long-acting restructuring that the present invention adopts mammalian cell to produce the coupling of non-polyoxyethylene glycol chemistry, rHuPH20 body inner blood Increased Plasma Half-life can be made, thus be applied to oncotherapy.PH20-HSA and PH20-IgFc mammalian cell of the present invention can adopt the specific method purifying of purifying HSA and IgFc after producing, and is better than the method that polyoxyethylene glycol chemistry coupling rHuPH20 produces long-acting people's Unidasa in production method and production prices.In addition, HSA and IgFc is people's source protein, and in body, security is better than polyoxyethylene glycol.
The above; it is only preferred embodiment of the present invention; not do any pro forma restriction to the present invention, those skilled in the art utilize the technology contents of above-mentioned announcement to make a little simple modification, equivalent variations or modification, all drop in protection scope of the present invention.
Claims (11)
1. a recombinant long-acting people Unidasa, is characterized in that, be the fusion rotein of people's Unidasa PH20 extracellular part and people's source protein, described people's source protein is human albumin HSA fragment or human normal immunoglobulin IgG2Fc fragment;
The fusion rotein of people's Unidasa PH20 extracellular part and human albumin HSA fragment is PH20-HSA, and its aminoacid sequence is as shown in SEQ ID No.4;
The fusion rotein of people's Unidasa PH20 extracellular part and human normal immunoglobulin IgG2Fc fragment is PH20-IgFc, and its aminoacid sequence is as shown in SEQ ID No.5.
2. an encoding gene for recombinant long-acting people Unidasa described in claim 1, is characterized in that, its gene order is as shown in SEQ ID No.6 or SEQ ID No.7.
3. an expression vector, is characterized in that, containing gene according to claim 2.
4. a host cell, is characterized in that, containing gene according to claim 2.
5. the production method of people's Unidasa according to claim 1, it is characterized in that, use animal cell expression vectors pMH3, pMH4 or pMH5 expressing gene sequence SEQ ID No.6 or SEQ ID No.7 in China ground hamster cell CHO being rich in GC, then carry out separation and purification and get final product; The gene order of described expression vector pMH3, pMH4 or pMH5 is respectively as shown in SEQIDNo.8,9,10.
6. the production method of people's Unidasa according to claim 5, is characterized in that, described PH20-HSA uses ProteinA affinity purification method to carry out separation and purification; Described PH20-IgFc adopts the hydrophobic blue gluing method of ion series connection to carry out separation and purification.
7. a preparation, is characterized in that, stably stored is had the right the people's Unidasa described in requirement 1, and described preparation comprises PH20-HSA or PH20-IgFc of 150-1500IU, 10mM Na2HPO4,0.03%CaCl2,0.1%EDTA-Na2,145mM NaCl, 0.1%HSA, and pH value is 5.5-7.5.
8. a compound preparation, is characterized in that, comprises preparation and particular treatment medicine that stably stored according to claim 7 has people's Unidasa.
9. recombinant long-acting people Unidasa according to claim 1 is coordinating the application in administration, it is characterized in that, recombinant long-acting people hyaluronidase activity being delivered to the tissue of human albumin or human normal immunoglobulin combination, organ, position when not being degraded; Or by particular treatment useful for drug delivery to the tissue at disease place, organ or position.
10. IgFc and the HSA fragment of recombinant long-acting Unidasa according to claim 1 provides that chemical coupling diagnostic reagent is carried out in NOT-function site, photographic developer, medicine, treatment toxin, gene enter diseased tissue organ.
11. application of recombinant long-acting people Unidasa according to claim 1 in industrial production micromolecule hyaluronic acid, is characterized in that, described recombinant long-acting people Unidasa produces micromolecule hyaluronic acid as toolenzyme degraded macromole hyaluronic acid.
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Effective date of registration: 20180403 Address after: 266000 Jiangshan Town Industrial Park, Laixi City, Qingdao, Shandong Patentee after: Qingdao respect Biotechnology Co. Ltd. Address before: The Jianggan District Canal Road East of Hangzhou city in Zhejiang province. The new Garden District Patentee before: Hui Mizhou |