CN110527680A

CN110527680A - A kind of hyaluronate lyase and its gene and application

Info

Publication number: CN110527680A
Application number: CN201910842217.XA
Authority: CN
Inventors: 江晓路; 张京良
Original assignee: Qingdao Marine Biomedical Research Institute Co Ltd
Current assignee: Qingdao Marine Biomedical Research Institute Co Ltd
Priority date: 2018-11-06
Filing date: 2019-09-06
Publication date: 2019-12-03
Anticipated expiration: 2039-09-06
Also published as: CN110527680B

Abstract

The invention belongs to field of biotechnology, and in particular to a kind of hyaluronate lyase and its gene, the expression and application of recombinant vector and cell and the hyaluronate lyase containing the gene.A kind of hyaluronate lyase, the amino acid sequence of the hyaluronate lyase is as shown in SEQ ID NO.1.A kind of hyaluronate lyase gene, encoding hyaluronan lyases, sequence are sequence shown in SEQ ID NO.2 or the complementary series of SEQ ID NO.2.The present invention successfully realizes the heterologous high-efficiency activated expression of bacterial origin hyaluronate lyase using engineered strain (Escherichia coli or bacillus subtilis) expression system; fermentative activity, which is significantly higher than, to be had been reported; realize the large-scale production of bacterial hyaluronidase lyases; and overcome the deficiency that wild strain isolates and purifies process complexity; high-purity hyaluronate lyase product is prepared, is laid the foundation for the industrialized production and hyaluronate lyase of hyaluronate lyase in the research application of biochemical industry and medicine and other fields.

Description

A kind of hyaluronate lyase and its gene and application

Technical field

The invention belongs to field of biotechnology, and in particular to a kind of hyaluronate lyase and its gene and application.

Background technique

Hyaluronic acid is a kind of chain high molecular polymer being alternately formed by connecting by acetylglucosamine glucuronic acid, It is the main constituents of animal tissue cell's epimatrix, is widely present in the biological tissues such as skin, cartilage, joint, vitreum In.Hyaluronic acid is able to maintain moisture of 500 times more than own wt, forms very sticky solution, is filled in extracellular Within substrates collagen fiber skeleton, the physical barriers formed between tissue cause certain biography in the transmission process of drug Matter resistance is the major obstacle of many drug deliveries.

Hyaluronidase is called hyaluronidase, is the glycosidase that can make hyaluronic acid that degraded effect occur, can also drop The acid mucopolysaccharide in other body tissues is solved, is widely present in eucaryote and prokaryotes, in animal body internal reference With many important biological processes, such as cell division, intercellular connection, the activity of reproduction cell, the transfection of DNA, embryo Fetal hair educates, the reparation of wounded tissue, is a kind of important physiological activator.And in 1928 by Duran Reynals It finds for the first time and is known as " invasin ", hyaluronidase was officially named by Chain and Duthie in 1940.It is transparent Matter acid enzyme is divided into 3 major class: (1) first kind is endo-beta-N-acetyl glucosidase (EC3.2.1.35), is mainly derived from The venom of vertebrate and certain biologies.This fermentoid is that hydrolase and transglycosylase are dropped by acting on β-Isosorbide-5-Nitrae glycosidic bond Solution substrate obtains the final product based on tetrose.(2) second classes are inscribe-beta-glucuronidase enzymes (EC3.2.1.36), main It to be extracted from the salivary gland and hookworm of leech.The fermentoid is hydrolase, and main function domain β -1,3 glycosidic bonds can be special Property degradation HA, generate final product based on tetrose or hexose.(3) third class is hyaluronate lyase (EC4.2.2.1), main If being generated by bacterium, β-l is acted on, 4 glycosidic bonds obtain 4,5- unsaturation disaccharide by β-cancellation mechanism.

Hyaluronidase reduces the viscosity of hyaluronic acid, and then reduce extracellular base by the hydrolysis of catalysis hyaluronic acid The viscosity of matter improves the penetrating power of liquid in tissue, improves the tissue permeability of drug, promotes drug diffusion and transmitting.Mesh Proinvasin has been used for multiple use, such as be used in the field of medicine hypodermoclysis, periocular surgery anesthesia, chemical drug and The absorption of contrast agent, treatment oedema, arthritis etc. in biological medicament rush infiltration, urography.

Recently as going deep into for hyaluronidase research and development, the potential important use of hyaluronidase: with it Its drug cooperates with medication, for promoting the diffusion absorption of subcutaneous medicament, synergistic antitumor, improveing marketed products etc., for weight The treatment of big disease especially tumour, presents very big potentiality.

Hyaluronidase and immunoglobulin combination are used for the treatment of primary immunodeficiency syndrome, in Europe, beauty State and Australia's listing, and current also multinomial hyaluronidase proceeds to different clinical ranks from immunoglobulin combination Section；A variety of drug combinations such as hyaluronidase and insulin, for diabetes, hereditary angioedema, hypercholesterolemia and The treatment of Huppert's disease.

It is many solid tumors (cancer of pancreas, prostate cancer, mammary gland that extrtacellular matrix deposition, which increases (expression of hyaluronic acid height), Cancer, oophoroma, film gland cancer, colon cancer, gastric cancer and non-small cell lung cancer etc.) typical speciality, hyaluronic acid and other components Protection network is generated around carcinoma cells with the enrichment of anomalous mode and is constituted support to it, hyaluronic acid and other components Pathologic accumulation can make the pressure increase of the interstitial fluid of tumour, tumor vessel compression, form one and be more conducive to growth of cancer cells Unique microenvironment promotes growth of cancer cells, and anti-tumor drug is hindered to diffuse to tumour cell, greatly limits most of anticancers The potential effect of drug, results in the resistance of therapeutic agent.

Target extracellular matrix in recent years combines the growth of tumour cell inhibitor of specificity, becomes the new for the treatment of tumour Therapy.Using hyaluronidase fast degradation hyaluronic acid, medicament transport impedance is released, and then anticancer therapeutic agent can be enable Tumor targets are more effectively transported to, its therapeutic effect is increased.Hyaluronidase and gemcitabine & taxol are combined, and are used for The treatment of cancer of pancreas；Hyaluronidase and methanesulfonic acid Ai Ruibulin are combined, for the treatment of metastatic breast cancer, at present into Row is clinical to II；Hyaluronidase modifier and PD-1 inhibitor are combined, and the treatment etc. for non-small cell lung cancer is made To different clinical stages.

Hyaluronidase can also be used in marketed products improvement, change marketed drug administration route.Such as by Herceptin (Herceptin) and MabThera (Rituximab) intravenous formulations are changed to subcutaneous injection formulation, treatment time It was shortened to from 2 ~ 3 hours 5 minutes, hence it is evident that shorten treatment time.Herceptin and MabThera subcutaneous injection formulation It has gone through to list in Europe at 2013 and 2014.Hyaluronidase and immunoglobulin are combined preparation HyQvia, are The first granted mensal subcutaneous injection immunoglobulin for primary immunodeficiency syndrome treatment.It monthly only needs subcutaneously to infuse It penetrates once, and whole therapeutic doses can be delivered in single injection site.For be reluctant repeatedly frequently be subcutaneously injected and instil Patient provides new medication selection.The drug is listed in May, 2013 in European Union at present, and in September, 2014 lists in the U.S..

In terms of medical cosmetology, cosmetics and skin care, hyaluronic acid filler may occur from slightly to serious early stage And late complication.Hyaluronidase can treat allergic reaction, blood vessel embolism as caused by filler, deficiency of straightening crooked, straighten crooked Excessively, the adverse reactions such as subcutaneous induration, delayed pigmentation, Immunoreactivity and granuloma, and significantly improve cellulite Present in fibrotic conditions.

Hyaluronidase used at present is mostly animal tissue source, belongs to hyaluronic acid hydrolysis enzyme.Up to now, The hyaluronic acid enzyme product of several animal origins of U.S. FDA approved is used for human medicine, but extracted due to animal tissue Hyaluronidase purity is low, and impurity is more, and anaphylaxis is stronger, and immunogenicity is strong and has potential propagation animal spongiform encephalopathy Risk, animal tissue extract hyaluronidase using production is very limited.Wherein Hyase and Wydase have been moved back City, Vitrase (Testis Caprae seu Ovis extraction) and Amphadase (bull testis extraction) also experienced the process of delisting/listing suspension, Product is purified be refining to obtain high purity product and supplementary data after just continue listing, price is high.

In the research of hyaluronidase recombinase, saccharomycete, Escherichia coli and bacillus subtilis all can be used for weight The host of group hyaluronidase.The hyaluronic acids such as leech hyaluronidase, people's hyaluronidase and streptomycete are had been achieved at present The recombinant expression of enzyme, bacterial origin hyaluronidase recombinant expression are less.Open source information display recombination fermenting and producing hyaluronic acid Production of enzyme is apparently higher than other techniques, and purifying process is fairly simple, and product purity is high, has good biochemical industry and doctor Medicine application prospect.

A kind of method of high efficiency recombinant expressed leech hyaluronidase is disclosed in 103614352 B of patent CN, not only It significantly improves yield and realizes the secreting, expressing (21333.33U/mL) of leech hyaluronidase, and simplify purifying step Suddenly.A kind of method using Pichia pastoris recombinant expression leech hyaluronidase is disclosed in 103695448 B of patent CN, And isolated and purified, it provides the foundation for the industrial applications of leech hyaluronidase.104745553 B of patent CN is public A kind of recombined human hyaluronidase and preparation method thereof has been opened, people's hyaluronidase gene integration is entered into Pichia yeast engineering In, it is purified by fermentation, obtains the recombined human hyaluronidase of a large amount of high activity high-purities.In 104342420 B of patent CN and 1942588 B of patent CN discloses a kind of utilization Chinese hamster ovary (CHO) cell recombinant expression of soluble people's hyaluronic acid The method of enzyme rHuPH20 effectively prevents the danger of animal spongiform encephalopathy, but since production process is complex and costly, and Half-life in vivo is shorter, influences therapeutic effect, it is difficult to meet hyaluronidase in medicine and industrial application.Patent CN It is disclosed in 105473607 A of 102439144 B and CN a kind of from streptomycetekoganeiensisHyaluronidase and Preparation method.103255076 B of patent CN disclose it is a kind of using bacillus hyaluronidase and preparation method thereof, with And the hyaluronidase is preparing the purposes in oligomerization hyaluronic acid or its salt.Su Kang etc., which discloses to derive from, bites nicotine section Bacillus (Artbrobacter nicotinovorous) hyaluronidase, molecular weight 22.1kDa, but highest producing enzyme vigor is only For 64.32U/mL, enzyme activity is lower, is unsuitable for scale development and application.Zhang Jingliang etc. patent " a kind of Arthrobacter globiformis and its The hyaluronidase of generation " (application number: 201610115793.0) discloses the Arthrobacter globiformis from intertidal zone sludge The hyaluronidase of A152, molecular weight 73.7kDa, regulating and controlling fermentation highest enzyme activity by alternating temperature is 7100U/mL.

Summary of the invention

Applicant of the present invention passes through in-depth study and creative labor, in view of the shortcomings of the prior art and insufficient, It provides a kind of hyaluronate lyase and its gene and application, the present invention also constructs the recombination of hyaluronate lyase gene Expression vector and the host cell for expressing hyaluronate lyase.Hyaluronate lyase fermentation liquid enzyme activity provided by the invention Power reaches as high as 4.9 × 10⁶U/mL is significantly higher than before more number reported enzyme activity in document.The enzyme has good temperature Stability and pH stability, product purity is high, prepares low molecule as promotion diffusant and biochemical industry application in terms of medicine There is unique advantage in terms of glycosaminoglycan.

According to the present invention, these purposes and the other purposes that can hereinafter become more apparent upon, are by following technical solution It realizes, specific technical solution is as follows: a kind of hyaluronate lyase, and amino acid sequence is as shown in SEQ ID NO. 1.

A kind of gene encoding the hyaluronate lyase, nucleotide sequence is as shown in SEQ ID NO.2 or SEQ The complementary series of ID NO.2.

A kind of recombinant vector, insertion contains above-mentioned hyaluronate lyase gene, according to the present invention preferably, institute on plasmid Selecting plasmid is escherichia coli plasmid pProEX-HTa or bacillus subtilis bacteria plasmid pHT43.

A kind of cell, the cell contain the hyaluronate lyase gene nucleotide series；Or the cell is by containing There is the inverted host cell of hyaluronate lyase gene nucleotide series recombinant vector and obtain, it is preferred that the place Chief cell is Escherichia coli or bacillus subtilis, further preferably e. coli bl21 (DE3) or bacillus subtilis WB800N。

It is separated to the bacterium HL6 of one plant of production hyaluronate lyase, from seawater with forward primer 27F:5 '- AGAGTTTGATCMTGCTCAG-3 ' (SEQ ID No:3), reversely draws 5 '-ACGGCTACCTTGTTACGACTT-3 ' (SEQ ID No:4), PCR amplification is carried out, 16S rRNA sequence is obtained as shown in SEQ ID NO. 5, by ncbi database 16S rRNA Sequence alignment identification, bacterial strain 16S rRNA length are 1443bp, have highest similarity 99.7% with arthrobacter bacteria, just Step is accredited as Arthrobacter.Morphology is carried out to the bacterial strain according to primary Jie Shi bacterium handbook and Physiology and biochemistry is identified, the results showed that, The bacterial strain is cultivated 48 hours at 24 DEG C, and bacterium colony is rounded, colorless and transparent.Gram-positive.It can use creatine, flesh ammonia As single carbon source, can liquefy gelatin and hydrolysis starch, but cannot restore NO for acid, glycine betaine and glycine₃To NO₂, need Biotin does not need ammonium sulfate, edaphic factor as growth factor as growth factor, leucine enzyme, glutamic acid enzyme positive, no Xylose, mannose, ribose and gossypose can be utilized.Morphology and Physiology and biochemistry the result shows that, the bacterial strain with application No. is 201610115793.0 invention in reported Arthrobacter globiformis A152 have significant difference.It is analyzed in conjunction with 16 rRNA sequences, Morphology and Physiology and biochemistry qualification result, by the Strain Designation be Arthrobacter globiformis (Arthrobacter globiformis) HL6。

Arthrobacter globiformis (Arthrobacter globiformis) HL6 is preserved in China typical culture collection center, Preservation date: on 07 05th, 2018, deposit number: CCTCC M 2018452.

A method of preparing the hyaluronate lyase, it is characterised in that comprise the steps of:

1) clone of hyaluronate lyase gene and analysis: extraction Arthrobacter globiformis (Arthrobacter globiformis) 2018452 genome of HL6 CCTCC M, according to the analysis of genome sequence and hyaluronate lyase functional gene, design is drawn Object obtains hyaluronate lyase gene by PCR using the genomic DNA of extraction as template；

2) building of hyaluronate lyase recombinant vector: by the nucleotide sequence of the hyaluronate lyase gene of acquisition through double It is connect after enzyme digestion with escherichia coli plasmid pProEX-HTa, obtains Escherichia coli recombinant vector, or by the hyaluronic acid of acquisition The nucleotide sequence of lyase gene is connect after double enzyme digestions with bacillus subtilis bacteria plasmid pHT43, obtains bacillus subtilis Bacterium recombinant vector；

3) hyaluronate lyase recombinant vector the building of hyaluronate lyase recombinant cell: is converted into e. coli bl21 (DE3) Escherichia coli hyaluronate lyase recombinant cell is obtained, or hyaluronate lyase recombinant vector is converted into withered grass bud Spore bacillus WB800N obtains bacillus subtilis hyaluronate lyase recombinant cell；

4) expression and purification of hyaluronate lyase: by the cell containing hyaluronate lyase gene or contain hyaluronic acid The cell inoculation of the recombinant vector of lyase gene is cultivated into bioreactor, inducing expression, collects expression product, is led to It crosses affinitive layer purification and obtains hyaluronate lyase activated protein, it optionally, can also be pure with gel filtration by ion exchange Change obtains hyaluronate lyase activated protein.

A kind of composition contains the hyaluronate lyase.The composition also contains in other food or drug Acceptable auxiliary material or carrier.Further the composition also contains or not contain pharmaceutically active agents.

Wherein the pharmaceutically active agents are selected from: insulin, antibody, chemotherapeutics, analgesic, anti-inflammatory agent, resists cell factor Bacteriocin, anti-amoeba worm agent, anti-Parkinson agent, anti-dysentery agent, Anticonvulsants, antidepressant, antirheumatic, antifungal agent, Rescinnamine, antihistaminic, α-adrenaline excitant, α blocking agent, anesthetic, bronchodilator, is killed livestock at antipyretic Agent, beta adrenergic blocking agent, calcium channel blocking agent, cardiovascalar agent, contraception medicament, decongestant agent, diuretics, calmness Agent, diagnostic reagent, electrolyte reagent, hypnosis medicament, hormone, muscular relaxation agent, flesh contracting agent, eye medicine, quasi- parasympathetic nerve Medicine, psychic energizer, tranquillizer, sympathetic transmitter releasers, uropoiesis agent, vagina medicinal agent, vitamin medicament, angiotensin-converter Any one of enzyme inhibitor and rush SLEEP AGENT.

The hyaluronate lyase gene or recombinant vector or recombinant cell or the Arthrobacter globiformis (Arthrobacter globiformis) HL6 CCTCC M 2018452 preparing the application on hyaluronate lyase.

The hyaluronate lyase or the composition are for treating and/or preventing disease or illness, or application In cosmetics or improve good appearance.

Hyaluronate lyase and its gene of the invention has the advantages that compared with prior art

The present invention successfully realizes bacterial origin hyalomitome using engineered strain (Escherichia coli or bacillus subtilis) expression system The heterologous high-efficiency activated expression of acid cleavage enzyme, fermentative activity are significantly higher than it has been reported that realizing bacterial hyaluronidase lyases Large-scale production, and the deficiency that wild strain isolates and purifies process complexity is overcome, prepare high-purity hyaluronate lyase Product, be hyaluronate lyase industrialized production and hyaluronate lyase biochemical industry and medicine and other fields research Using laying the foundation.

Detailed description of the invention

Fig. 1: Arthrobacter globiformis (Arthrobacter globiformils) HL6 CCTCC M 2018452 cell it is total Electrophorogram of the DNA in 1% Ago-Gel, left side marker, right side are sample；

Fig. 2: electrophorogram of the hyaluronate lyase gene in 1% Ago-Gel.Left side is marker, and right side is sample Product；

Fig. 3: the hyaluronate lyase SDS-PAGE electrophoresis spectrum of purifying.Left side is standard protein molecular weight, and right side is through pure The hyaluronate lyase destination protein of change；

Fig. 4: the viscosity variation of hyaluronate lyase degradation hyaluronic acid.Abscissa is the reaction time (s), and ordinate is viscosity (mPa ﹒ s)；

Fig. 5: influence of the temperature to hyaluronate lyase.Abscissa is different temperatures, and ordinate is opposite enzyme activity；

Fig. 6: hyaluronate lyase is in 4 DEG C and 37 DEG C of temperature stability.Abscissa is different time, and ordinate is opposite enzyme It is living；

Fig. 7: the pH influence to hyaluronate lyase.Abscissa is different pH, and ordinate is opposite enzyme activity；

Fig. 8: pH stability of the hyaluronate lyase in pH7.0.Abscissa is different time, and ordinate is opposite enzyme activity；

Fig. 9: influence of the hyaluronate lyase to taxol induced hela Apoptosis.Abscissa is sample and concentration, indulges and sits It is designated as cell survival rate (%).

Specific embodiment

The present invention realizes the recombinant expression of hyaluronate lyase, is prepared for enzyme activity height, high high-quality of product purity Recombination hyaluronate lyase large-scale production may be implemented according to the technique and scheme of the present invention in hyaluronic acid enzyme product.

As used herein, " hyaluronidase activity " is defined with the unit of activity of enzyme.The traditional Chinese medicines in Allusion quotation method (referring to 2015 editions general rules 1207 of Chinese Pharmacopoeia: hyaluronidase measuring method) measurement is in the fermentation liquid as made from above scheme Hyaluronic acid enzyme activity.Fermentation broth enzyme activity unit definition are as follows: every milliliter of fermentation liquid (U/mL) containing enzyme activity unit.

As used herein, " the plate transparent circle method " is operated by based on following principle: hyaluronic acid is Polymeric polyanion polysaccharide, the Precipitation in certain density aqueous surfactant solution, hydrolyzed low molecule are transparent Matter acid will not be deposited precipitation.Identification hyaluronidase activity that can be fast and convenient using this principle.

Hyaluronate lyase of the invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide.Polypeptide of the invention can To be native purified product or chemically synthesized product, or using recombinant technique from host (for example, colon bacteria and withered Careless bacillus) in generate.

The present invention includes the segment of hyaluronate lyase.As used herein, refer to be kept substantially it is of the invention natural The identical biological function of hyaluronidase or active polypeptide.Polypeptide fragment of the invention, which can be (i), one or more Conservative or non-conservative amino acid residue (preferably conservative amino acid) substituted polypeptide, and such substituted ammonia Base acid residue, which can be, may not be by genetic code encoding, or (ii) has in one or more amino acid residues The polypeptide of substituent group, or (iii) additional amino acid sequence are fused to this polypeptide sequence and polypeptide (such as leading sequence that is formed Column or secretion sequence or for purifying the sequence of this polypeptide or proprotein sequence or fusion protein).According to the definition of this paper these Segment, derivative and analogue belong to scope known to those skilled in the art.

The technical solution that present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are only used for Illustrate the present invention rather than limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually Such as J. Pehanorm Brooker is write according to normal conditions, Molecular Cloning:A Laboratory guide, the third edition, Science Press, in 2002 The condition, or according to the normal condition proposed by manufacturer.

The clone of 1. hyaluronate lyase gene of embodiment and analysis

Take culture to logarithmic phase Arthrobacter globiformis (Arthrobacter globiformis) HL6 CCTCC M 2018452 Bacterium solution, according to TIAamp Bacteria DNA Kit(TIANGEN BIOTECH(BEIJING) CO., LTD) kit operation Illustrate to carry out DNA extraction, using progress DNA band detection in 1% agarose gel electrophoresis, testing conditions: 5 μ L of applied sample amount, electricity 120V, electrophoresis 30min are pressed, observes band (electrophorogram is shown in attached drawing 1) in gel imaging system, band homogeneity is good.It will The DNA sample of extraction is sequenced.

With Arthrobacter globiformis (Arthrobacter globiformis) HL6 CCTCC M 2018452 total DNA be template, It is analyzed by the functional gene of database, hyaluronate lyase gene HyLs is expanded using primers F and R:

F:ATGTTCGCCAACCACGCCT (SEQ.ID.No.6)

R:GGATACCGGGCGACGTTAGC (SEQ.ID.No.7)

It is expanded using PCR, 50 μ l reaction systems are as follows:

Contain DNA profiling, 1 μ L in the reaction system of 50 μ L；F (10 μM), 1 μ L；R (10 μM), 1 μ L；DNTP (each 2 .5mM), 4 μ L；Taq (2U/ μ L), 1 μ L；10 × Taq buffer, 5 μ L；DdH2O adds to 50 μ L.

PCR amplification program are as follows: 95 DEG C of initial denaturations 5 min, 95 DEG C of denaturation 45s, 56 DEG C of annealing 45 s, 72 DEG C of 1 min of extension, Circulation 30 times, 72 DEG C of 10 min.(electrophoretogram is sequenced in PCR product after 1% agarose electrophoresis verifies as single specificity band Spectrum is shown in attached drawing 2).

Acquisition Arthrobacter globiformis (Arthrobacter globiformis) HL6 CCTCC M 2018452 hyaluronic acid split The expressing gene HyLs of enzyme is solved, 2298 bp of length, sequencing sequence is as shown in sequence table SEQ .ID.No.2.By SEQ.ID.No.2 Shown gene order is compared with whole genome sequence, and the two is consistent.The gene encodes the albumen being made of 765 amino acid Matter, sequence is as shown in sequence table SEQ .ID.No.1.Blast sequence in NCBI database analysis shows, which belongs to polysaccharide 8 family of lyases, amino acid sequence and registration hyaluronate lyase amino acid sequence similarity highest only up to 90.46% (Arthrobacter hyaluronate lyase: WP_039242887.1, WP_018778839.1) illustrates that the albumen is a new egg White, which is a new gene.

The building of 2. hyaluronate lyase Escherichia coli recombinant vector of embodiment

With Arthrobacter globiformis (Arthrobacter globiformis) HL6 CCTCC M 2018452 total DNA be template, pass through The functional gene of database is analyzed, and is split with primer EC-F and EC-R the amplification hyaluronic acid containing restriction enzyme digestion sites Solve enzyme gene HyLs(and be free of signal peptide):

EC-F:GGACTAGTCATGTTCGCCAACCACGCCT(SEQ.ID.No.8)

EC-R:GGGGTACCCGGATACCGGGCGACGTTAGC(SEQ.ID.No.9)

Wherein, ACTAGT is the restriction enzyme site of restriction endonuclease Spe I, and GGTACC is the restriction enzyme site of restriction endonuclease Kpn I.

It is expanded using PCR, 50 μ l reaction systems are as follows:

Contain DNA profiling, 1 μ L in the reaction system of 50 μ L；EC-F (10 μM), 1 μ L；EC-R (10 μM), 1 μ L；DNTP (each 2 .5mM), 4 μ L；Taq (2U/ μ L), 1 μ L；10 × Taq buffer, 5 μ L；DdH2O adds to 50 μ L.

PCR amplification program are as follows: 95 DEG C of initial denaturation 3 min, 96 DEG C of denaturation 30s, 60 DEG C of annealing 45 s, 70 DEG C of extension 90s are followed Ring 30 times, 70 DEG C of 10 min.

Shown in PCR product 1% agarose electrophoresis of progress and sequence verification, extension increasing sequence and sequence table SEQ .ID.No.2 thoroughly Bright matter lyase gene HyLs is consistent.PCR product is according to Cycle-Pure Kit(OMEGA Bio-Tek Co.) purified reagent The operating method that box requires is purified.

Carrier pProEX-HTa and PCR amplification gene are subjected to double enzyme digestions respectively, digestion products carry out agarose electrophoresis, According to Gel extraction kit(OMEGA Bio-Tek Co.) glue extracts kit require operating method recycled.

It will be connect, be contained with carrier pProEX-HTa containing hyaluronate lyase gene order segment with T4 ligase There is the recombinant vector HTa-HyLs of hyaluronate lyase gene.

Embodiment 3. recombinates expression of the hyaluronate lyase in Escherichia coli

Select coli strain DH5 α, by permissive cell preparation, heat-shock transformed (42 DEG C, 60s), be incubated for (37 DEG C, 160rpm, 45min), transformant is being screened containing 75 μ g/mL ampicillin sodium LB solid plates, not being converted bacterial strain can not In plated growth.It is detected through PCR, obtains positive colony bacterial strain, tried with Plasmid Mini Kit(OMEGA Bio-Tek Co.) Agent box carries out plasmid extraction, obtains recombinant plasmid HTa-HyLs.

Select E. coli expression strains BL21(DE3), by competent cell preparation, heat-shock transformed (42 DEG C, 60s), It is incubated for (37 DEG C, 45min), the recombinant plasmid HTa-HyLs after extraction is transformed into E. coli expression strains BL21(DE3), 75 The screening of μ g/mL ampicillin LB solid plate, 37 DEG C of culture 16h obtain transformant, and picking individual colonies transformant PCR is detected, Obtain positive colony bacterial strain.Glycerol at -80 DEG C is placed in save.

Positive colony bacterial strain is inoculated in LB liquid medium (75 μ g/mL ampicillin sodium), 37 DEG C of cultures are extremely OD₆₀₀When being 0.6 ~ 0.7, be added IPTG to final concentration of 0.25mM, 24 DEG C, 180r/min Fiber differentiation for 24 hours, by fermentation liquid 4 At DEG C, 8000r/min is centrifuged 10min, collects fermented supernatant fluid, and measuring extracellular hyaluronic acid enzyme activity is 8.5 × 10⁶U/mL。

The construction and expression of 4. hyaluronate lyase bacillus subtilis recombinant vector of embodiment

With Arthrobacter globiformis (Arthrobacter globiformis) HL6 CCTCC M 2018452 total DNA be template, pass through The functional gene of database is analyzed, and is split with primer BS-F and BS-R the amplification hyaluronic acid containing restriction enzyme digestion sites Solve enzyme gene HyLs(and be free of signal peptide):

BS-F:TGCTCTAGAGCAATGTTCGCCAACCACGCCT(SEQ.ID.No.10)

BS-R:CGCGACGTCCGCGGATACCGGGCGACGTTAGC(SEQ.ID.No.11)

Wherein, TCTAGA is the restriction enzyme site of restriction endonuclease Xba I, and GACGTC is the restriction enzyme site of restriction endonuclease AatII.

It is expanded using PCR, 50 μ l reaction systems are as follows:

Contain DNA profiling, 1 μ L in the reaction system of 50 μ L；BS-F (10 μM), 1 μ L；BS-R (10 μM), 1 μ L；DNTP (each 2 .5mM), 4 μ L；Taq (2U/ μ L), 1 μ L；10 × Taq buffer, 5 μ L；DdH2O adds to 50 μ L.

PCR amplification program are as follows: 95 DEG C of 3 min of initial denaturation, 96 DEG C of denaturation 30s, 60 DEG C of 45 s of annealing, 70 DEG C extend 90s is recycled 30 times, 70 DEG C of 10 min.

Carrier pHT43 and PCR amplification gene are subjected to double enzyme digestions respectively, digestion products carry out agarose electrophoresis, according to Gel extraction kit(OMEGA Bio-Tek Co.) glue extracts kit require operating method recycled.Use T4 Ligase will be connect containing hyaluronate lyase gene order segment with carrier pHT43, and acquisition contains hyaluronate lyase The recombinant vector pHT43-HyLs of gene.

Select coli strain DH5 α, by permissive cell preparation, heat-shock transformed (42 DEG C, 60s), be incubated for (37 DEG C, 160rpm, 45min), transformant is being screened containing 75 μ g/mL ampicillin sodium LB solid plates, not being converted bacterial strain can not In plated growth.It is detected through PCR, positive colony bacterial strain is obtained, according to Plasmid Mini Kit(OMEGA Bio-Tek Co.) The method that kit requires carries out plasmid extraction, obtains recombinant plasmid pHT43-HyLs.By the recombinant plasmid pHT43- after extraction HyLs is transformed into bacillus subtilis expression bacterial strain WB800N, the screening of 75 μ g/mL ampicillin LB solid plates, 37 DEG C of trainings It supports 16h and obtains transformant, picking individual colonies transformant PCR detection obtains positive colony bacterial strain.Glycerol at -80 DEG C is placed in save.

Positive colony bacterial strain is inoculated in LB liquid medium (75 μ g/mL ampicillin sodium), 37 DEG C of cultures are extremely OD₆₀₀When being 0.6 ~ 0.7, be added IPTG to final concentration of 0.25mM, 24 DEG C, 180r/min Fiber differentiation for 24 hours, by fermentation liquid 4 At DEG C, 8000r/min is centrifuged 10min, collects fermented supernatant fluid, and measuring extracellular hyaluronic acid enzyme activity is 5.3 × 10⁵U/mL, Fermentation broth enzyme activity is lower than Escherichia coli fermentation expression.

5. Arthrobacter globiformis of embodiment (Arthrobacter globiformis) fermentation of HL6 CCTCC M 2018452 Produce hyaluronate lyase

By the Arthrobacter globiformis (Arthrobacter globiformis) HL6 CCTCC M 2018452 strain inoculated arrive In sterilizing seed culture medium (seed culture based formulas are as follows: 0.2 g/L of Sodium Hyaluronate, 5 g/L of glucose, 2 g/L of peptone, Dipotassium hydrogen phosphate 1.5 g/L, MgSO₄0.5 g/L, pH 7.5), 28 DEG C, 100 r/min, 12h is cultivated, obtains seed liquor.

Seed culture fluid is inoculated into the fermentation medium of sterilizing (2 g/L of Sodium Hyaluronate, 10 g/L of glucose, egg White 5 g/L of peptone, dipotassium hydrogen phosphate 1.5 g/L, MgSO₄0.5 g/L, pH 7.5), 32 DEG C, 200 r/min cultivate 72 h, obtain Fermentation liquid containing hyaluronidase, by fermentation liquid at 4 DEG C, 8000r/min is centrifuged 10min, collects fermented supernatant fluid, measures born of the same parents Outer hyaluronic acid enzyme activity is 2 × 10⁵U/mL has reported enzyme activity higher than majority.

Embodiment 6. recombinates enzyme purification

1) fermented supernatant fluid after being centrifuged embodiment 3 carries out affinity chromatography (GE company) with nickel column first, according to ni-sepharose purification Specification carries out initial gross separation purifying, collects pure initial gross separation purified product；

2) step 1) preliminary purification product is used into QFF-Sepharose again^®Fast Flow chromatographic column (GE company) purifies again, First use 20mM, the Tris-HCl buffer of pH7.5 balances pillar, with the 20mM for containing concentration being 0-1mol/L sodium chloride after loading Tris-HCl (pH 7.5) buffer carries out linear gradient elution, and protein peak is detected at 280nm, and component is collected by pipe, measurement The hyaluronic acid enzyme activity of each pipe collection liquid collects active albumen fraction；

3) the activated protein fraction for further collecting step 2 is purified with Sephadex G100 gel (GE), and eluent is 7.0 phosphate buffer of 20mM pH is collected by pipe, detects hyaluronic acid enzyme activity, collecting active albumen fraction is Hyaluronate lyase (the Rate activity: 5.1 × 10 of purifying⁸U/mg).Purification of hyaluronic acid enzyme is subjected to SDS-PAGE electrophoresis, or It obtains the single band of albumen (electrophorogram is shown in attached drawing 3), molecular weight is about 80 kDa, and saturating according to sequence table SEQ .ID.No.2 The hyaluronate lyase sequence SEQ.ID.No.1 result that bright matter lyase gene speculates is almost the same.

7. hyaluronate lyase of embodiment is reducing the application in hyaluronic acid viscosity

According to the method for European Pharmacopoeia (EP 9.0), by the hyaluronidase sodium chloride solution (9g/L) and 1 of 100 U/mL of 1mL The hyaluronic acid of mL10g/L mixes at 20 DEG C, detects in solution 90s stick at rheometer (Rhemeter MCR 301) rapidly Spend consecutive variations, not have the group of enzyme hyaluronic acid solution as blank control, with addition 100 DEG C at heat 30 minutes handle after The group for being cooled to 20 DEG C of enzyme solution is negative control, using Zhong Jian institute hyaluronidase standard items as positive control.Experimental result is shown in attached Fig. 4, the results showed that sample prepared by 3-embodiment of embodiment 6 can be such that hyaluronic acid viscosity declines rapidly in a short time, With hyaluronidase standard items hyaluronidase activity having the same.Show prepared by the hyaluronidase of this project preparation simultaneously Low molecular weight hyaluronic acid and degradation extracellular matrix hyaluronic acid, promote drug diffusion etc. to have potential uses.

Influence of 8. temperature of embodiment to hyaluronate lyase

Respectively at 20,30,37,40,50,60 DEG C, prepared according to Chinese Pharmacopoeia prescriptive procedure measurement 3-embodiment of embodiment 6 Sample hyaluronidase enzyme activity, figure is done with opposite enzyme activity and compares (highest enzyme activity be 100%).Experimental result is shown in attached drawings 5, real Test the result shows that 3-embodiment of embodiment 6 prepare hyaluron sample within the temperature range of 30-50 DEG C have it is good Enzyme activity, optimal reactive temperature are 37 DEG C.

The sample of 3-embodiment of embodiment 6 is stored 6 hours at 4 DEG C and 37 DEG C, it is primary every sampling in 1 hour, according to Chinese Pharmacopoeia regulation enzyme activity determination method measurement each sample enzyme activity is done with investigating the temperature stability of each sample with opposite enzyme activity Figure compares (highest enzyme activity is 100%).Experimental result is shown in attached drawings 6, the experimental results showed that the preparation of 3-embodiment of embodiment 6 is transparent Matter acid enzyme sample is stablized at 4 DEG C, stores 6 hours enzyme activity and varies less, and still contains 95% or more of initial enzyme activity, and become In stabilization.At 37 DEG C then each sample is tended towards stability 10% or so the decline of the 1st hour enzyme activity is more, slowly under Drop, stores 6 hours at 37 DEG C and still retains 80% or more of initial enzyme activity, has good temperature stability.

Influence of 9. pH of embodiment to hyaluronate lyase

Hyaluronic acid substrate is prepared according to pharmacopoeial requirements using pH 3-10 purified water respectively, then measures 3-embodiment of embodiment The sample of 6 preparations hyaluronidase enzyme activity at different pH does figure with opposite enzyme activity and compares (highest enzyme activity is 100%).Experiment knot Fruit sees attached drawing 7, the experimental results showed that hyaluron sample optimal pH prepared by 3-embodiment of embodiment 6 is 7.0, In Good enzyme activity (opposite enzyme activity >=80%), the pH of higher (>=pH10) and lower (pH≤4) are all had in the range of pH6-8 The enzyme activity of hyaluronidase can be completely inhibited.

By the sample of the preparation of 3-embodiment of embodiment 6 in pH 7.0(room temperature) under the conditions of store 6 hours, every 1 hour Sampling is primary, measures each sample enzyme activity according to Chinese Pharmacopoeia regulation enzyme activity determination method, to investigate the pH stability of each sample, with Opposite enzyme activity does figure and compares (highest enzyme activity is 100%).Experimental result is shown in attached drawings 8, the experimental results showed that 3-embodiment of embodiment 6 The hyaluron sample of preparation is relatively more in enzyme activity decline in the 1st hour under optimal pH 7.0 (room temperature), decline about 10%, Then it can remain more stable, slowly decline, still there is 80% or more opposite enzyme activity at 6 hours, it is more stable, it was demonstrated that this case The hyaluron sample of preparation has good pH stability.8 temperature stability data in conjunction with the embodiments, show this case system Standby hyaluronate lyase sample, either Escherichia coli recombination fermentation, bacillus subtilis recombination fermentation, wild strain Fermentation and recombination fermentation purifying enzyme product, not only yield is high, and enzyme activity is high, and property is stablized, and identical most thermophilic is all had Degree and pH, and all have good temperature and pH stability, biological medicine and in terms of there is good open Send out application prospect.

Influence of 10 hyaluronate lyase of embodiment to the apoptosis of taxol induced human cervical carcinoma cell (Hela cell)

By Hela cell with 1 × 10⁴/ hole is inoculated with 96 orifice plates, and drug to be measured is added after cell is adherent, and (taxol & hyaluronic acid is split Solve enzyme (purifying of embodiment 6 preparation)).It is trained in the DMEM containing 10% calf serum, 100U/mL penicillin and 100ug/mL streptomysin It supports in base, in 37 DEG C, 5% CO₂It is cultivated 48 hours under saturated humidity.Mtt assay is used after culture, is measured under wavelength in 570nm Detect each hole absorbance.The influence of drug cell proliferation is calculated according to each hole absorbance, calculates cell survival rate formula: cell Survival rate (%)=drug containing hole OD average value/control wells OD average value * 100, inhibiting rate=1- survival rate.Experimental result is shown in attached drawing 9, According to the experimental results, when paclitaxel concentration is 0.01uM, compared with blank control group, the growth of the concentration versus cell is basic There is no inhibiting effect, adds the hyaluronate lyase of 1U/mL, 10U/mL and 100U/mL respectively, it is also basic to the growth of cell There is no inhibiting effect, illustrates hyaluronidase itself to the no inhibiting effect of the growth of hela cell.When addition paclitaxel concentration When for 0.1uM, taxol produces apparent inhibiting effect to the growth of hela cell, and hela cell survival rate is about 76.5%, After adding the hyaluronate lyase of various concentration, taxol to the inhibiting effect of hela cell with additive amount increase by Cumulative strong, survival rate is substantially reduced, and when adding 10U/mL, the survival rate of hela cell is about 65.5%, is continued raising concentration and is arrived When 100 U/mL, survival rate is about 61.2%, and taxol and hyaluronate lyase mention the inhibiting rate of hela cell from 23.5% It is raised to 38.8%, hence it is evident that promote the inhibiting effect of growth of the taxol to hela cell.Reason may be hyaluronate lyase The high concentration hyaluronic acid in tumour cell microenvironment is degraded, the easier arrival tumor cell surface of taxol is allow, And then play drug effect.

The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although referring to aforementioned reality Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation The technical solution that example is stated is modified or equivalent replacement of some of the technical features；And these are modified or replace It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.

Sequence table

<110>limited liability company, Qingdao Haiyang biological medicine research institute

<120>a kind of hyaluronate lyase and its gene and application

<150> 2018113109388

<151> 2018-11-06

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 765

<212> PRT

<213> Arthrobacter globiformis

<400> 1

Met Pro Ala Ala His Ala Thr Ala Ala Ala Gly Pro Gly Thr Ala Gly

1 5 10 15

Pro Ala Ala Leu Ala Ser Ala Thr Val Ala Gly Ile Thr Gly Ala Ala

20 25 30

Val Ile Gly Ala Gly Ala Pro Ala Pro Ala Ala Ala Val Thr Ala Leu

35 40 45

Ala Thr Leu Ala Ala Ala Ser Leu Ala Leu Leu Ala Ala Val Ser Gly

50 55 60

Ala Thr Ser Val Pro Thr Ala Leu Ser Pro Ala Leu Ala Ala Gly Met

65 70 75 80

Val Thr Thr Thr Thr Ala Leu Ser Gly Leu Ala Ala Ala Thr Ala Thr

85 90 95

Pro Thr Ala Ala Val Pro Gly Ala Ser Ala Val Leu Ala Ala Ile Leu

100 105 110

Ala Gly Leu Ala Ala Ala Ala Thr Leu Cys Thr His Ala Gly Ala Gly

115 120 125

Gly Val Gly Ala Thr Thr Ser Thr Gly Ile Gly Val Pro Ala Ala Leu

130 135 140

Ala Ala Ala Met Val Leu Leu His Ala Gly Leu Ser Ala Ala Gly Ile

145 150 155 160

Gly Ala Thr Ser Ala Ala Ile Ala His Pro Val Pro Ala Pro Thr Leu

165 170 175

Gly Pro Pro Pro Leu Ala Gly Leu Ile Thr Ser Val Gly Ala Ala Ala

180 185 190

Val Ala Leu Cys Gly Gly Val Ile Ile Ala Ser Leu Ala Gly Gly Ala

195 200 205

Pro Gly Leu Leu Ala His Ala Val Ala Gly Leu Ser Gly Val Thr Gly

210 215 220

Thr Val Thr Ser Gly Ala Gly Ile Pro Ala Ala Gly Ser Pro Ile Gly

225 230 235 240

His Ser Thr Thr Pro Thr Thr Gly Ser Thr Gly Val Val Leu Leu Thr

245 250 255

Gly Leu Ser Leu Leu Pro Ser Leu Leu Gly Gly Thr Ala Pro Gly Val

260 265 270

Ser Ala Pro Ser Ala Ser Ile Pro Pro Ala Ala Val Gly Gly Ser Pro

275 280 285

Ala Pro Val Met Ile Ala Gly Ala Met Ala Ala Ser Val Ala Gly Ala

290 295 300

Ser Ile Ser Ala Gly Ala Ala Thr Gly Thr Ala Leu Gly Ala Ser Ala

305 310 315 320

Ile Gly Ala Ile Leu Leu Leu Ala Ala Ala Met Ala Pro Ala Thr Ala

325 330 335

Thr Ala Thr Ala Gly Leu Cys Ala Gly Thr Ile Ala Ala Ala Thr Thr

340 345 350

Ala Pro Ile Leu Ala Gly Ala Ser Leu Pro Ala Thr Ala Leu Val Leu

355 360 365

Gly Leu Gly Ser Thr Gly Ile Ala Pro Val Ala Gly Ala Pro Gly His

370 375 380

Ala Leu Pro Pro Ala Met Ala Ala Thr Met His Ala Gly Pro Gly Thr

385 390 395 400

Ala Leu Ser Leu Ser Leu Ser Ser Ala Ala Ile Ala Thr Thr Gly Cys

405 410 415

Gly Ala Gly Gly Ala Ala Ala Gly Thr His Thr Gly Ser Gly Met Thr

420 425 430

Thr Pro Thr Thr Ser Ala Leu Gly Gly Thr Ala Ala Ala Pro Thr Ala

435 440 445

Thr Ala Ala Thr Ala Ala Leu Pro Gly Ile Thr Val Ala Thr Thr Pro

450 455 460

Leu Pro Ala Leu Val Gly Gly Gly Thr Gly Ala Ala Val Pro Ala Ala

465 470 475 480

Gly Thr Ser Gly Ala Thr Ala Leu Gly Gly Val Ala Ala Val Gly Gly

485 490 495

His Leu Val Gly Pro Gly Ala Thr Gly Leu Ser Ala Ala Leu Ser Thr

500 505 510

Pro Val Ser Gly Gly Ala Thr Val Cys Leu Gly Ala Ala Ile Thr Thr

515 520 525

Gly Ser Gly Ala Ala Val Gly Ser Ile Val Ala His Ala Ala Leu His

530 535 540

Gly Gly Ser Ala Thr Leu Thr Thr Ala Ala Gly Thr Ile Ala Gly Ser

545 550 555 560

Val Gly Ser Ala Gly Val Leu Ser Gly Gly Ala Thr Val His Leu Gly

565 570 575

Gly Pro Gly Gly Thr Ala Met Leu Ala Ala Ser Pro Leu His Val Leu

580 585 590

Ala Gly Thr Ala Ser Gly Ser Thr Ser Gly Val Ala Thr Ala Gly Ser

595 600 605

Thr Thr Val His Gly Ala Thr Pro Ala Thr Leu Thr Val Ala His Gly

610 615 620

Ala Gly Pro Ala Ala Gly Ser Thr Ala Thr Val Val Ala Pro Gly Ala

625 630 635 640

Ser Val Ala Leu Thr Ala Leu Leu Val Gly Gly Ala Leu Thr Ala Val

645 650 655

Ile Ala Ala Ala Thr Thr Ala Gly Ser Val Gly Pro Leu Ala Ser Leu

660 665 670

Thr Thr Ala Ala Thr Pro Thr Leu Pro Gly Met Ala Gly Ala Leu Gly

675 680 685

Ala Ser Gly Pro Ala Cys Val Val Pro Ser Ala His Gly Ala Gly Leu

690 695 700

Ser Leu Ala Pro Ser Gly Pro Thr Gly Leu Ala Ala Ser Leu Thr Leu

705 710 715 720

Thr Leu Pro Gly Gly Thr Thr Ser Ser Val Leu Gly Gly Thr Gly Thr

725 730 735

Leu Gly Thr Ala Ala Ala Gly Ala Ser Thr Val Thr Leu Ala Thr Ala

740 745 750

Gly Leu Ala Gly Gly Thr Leu Val Ile Thr Leu Ala Ala

755 760 765

<210> 2

<211> 2298

<212> DNA

<213> Arthrobacter globiformis

<400> 2

atgttcgcca accacgcctg ggccgacgcg gaaccgggca ccgcggagtt cgcagcgctg 60

cgaagccgtt gggtggacca gatcacgggc cgcaacgtca tccaagccgg cgatccggac 120

tttgccaggg cggtgacagc gctgaacacc aaagccgctg actccttggc aaagctcaac 180

cgggtttcag gccgaacctc ggtctttacg gacttgtcct tcgccaagga tgcagagatg 240

gtcaccacgt acacgcgttt atcccagctc gctgctgcct gggcaacacc aacggccgcg 300

gtgtttggtg attccgcagt actggcagac atcaaggcgg gcctcgccga cgccaatacc 360

ctctgctacc acgctggcag ggaagaggtc ggcaactggt ggtcgtggga aatcggtgtg 420

ccccgtgcct tggccgacgc catggtgctt cttcacgccg agctgtccgc cgctgaaata 480

caggcctaca gtgcggcgat cgaccatttt gtgccggacc cttggctgca gttcccaccc 540

aagcgcggca agatcacctc cgtgggcgcc aaccgtgtgg acctgtgcca aggggtcatc 600

atccggtccc tcgctggaga agatccgggc aagctcaacc acgcagtcgc cggactcagc 660

caggtgtggc agtacgtcac cagtggtgac ggaatcttcc gggacggctc gtttatccaa 720

cacagcacca ccccgtacac gggctcctac ggggtggtcc tgctcaccgg attgtccaag 780

ttgttctccc tcctgggagg cacggcgttc gaagtttcgg acccctcgcg cagtattttc 840

ttcgacgcag tggagggttc gtttgcgccc gtcatgatca acggggccat ggccgattcc 900

gtgcgcggca ggagtatcag ccgcgaggcc aacaccggct acgacctggg ggcatcggcc 960

atcgaagcca ttctgctgct ggcccgggcc atggatccag ctactgccac acgatggaga 1020

gggctgtgcg cgggatggat tgcgcgcaat acgtaccggc ccatcctcgc aggggccagc 1080

ctgcccagga ctgcattggt gaaggagctt cagtcaacgg gtatcgcacc ggtggcagaa 1140

gcccccgggc acaggctctt ccctgcgatg gaccgcacca tgcaccgggg acccggctgg 1200

gcattgtcgc tctccctgtc cagcaaccgc atcgcctggt acgaatgcgg caatggcgag 1260

aacaaccgcg gctatcacac gggttccggc atgacgtact tctatacgtc cgatctcggc 1320

caatacgatg acgcgttctg ggccacagcc aactacaacc gccttccggg catcaccgtg 1380

gacaccactc cgttgccgga caaggtggag ggtgaatggg gtgccgccgt tcctgcgaat 1440

gaatggagtg gcgccacggc gcttggcggg gttgccgccg tcggacaaca cctggtggga 1500

ccgggccgca cgggcctgtc cgccaggaag tcctggtttg tcagcggcga ggccactgtc 1560

tgcctcggcg ccgacatcac cactggttcc ggggccaggg tggaaagcat cgttgaccac 1620

cgcaacctcc accagggcag caatacactc acgacggcgg caggcaccat cgccggatcg 1680

gtcggcagtg ctgaggtact gagcgaagaa cgctgggttc atttggaggg tttcggaggc 1740

tacgccatgc tggacgattc cccgcttcac gtgctccggg aaacccgatc aggcagctgg 1800

tccggggtca acaccaacgg cagcaccacc gtccaccagc gcacctttgc caccctctac 1860

gtagaccacg gcgccggacc tgctgcgggc agctatgcct atgtggttgc tccgggcgct 1920

tctgtgaacc tgacccggaa gctggtgcag ggggacaaat accgggtgat ccgcaacgat 1980

acaacggcac agtccgtgga gttcaaggca tcgaagacca cggcagcaac cttctggaag 2040

cccgggatgg cgggggatct gggtgcgtcc gggcctgctt gcgtggtgtt ctccaggcac 2100

ggaaatgagt tgagcctggc gttcagtgag ccaacgcaga aggctgccag cctcacgctg 2160

accctgcccc agggcacatg gtccagcgtg ctggaaggca cgggcacact ggggaccgac 2220

gcagacggcc ggagtacggt gacccttgat acggccggcc tgaatggcca gacgaaggtc 2280

atcacactgc ggcgctaa 2298

<210> 3

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

agagtttgat cmtgctcag 19

<210> 4

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

acggctacct tgttacgact t 21

<210> 5

<211> 1443

<212> DNA

<213> Arthrobacter globiformis

<400> 5

tttttagagt ttttttattc gtggctcagg atgaacgctg gcggcgtgct tcacacatgc 60

aagtcgaacg atgatcccag cttgmtgggg gattagtggc gaacgggtga gtaacacgtg 120

agtaacctgc ccttgactct gggataagcc tgggaaactg ggtctaatac cggatatgac 180

catctgacgc atgtcatggt ggtggaaagc ttttgtggtt ttggatggac tcgcggccta 240

tcagcttgtt ggtggggtaa tggcctacca aggcgacgac gggtagccgg cctgagaggg 300

tgaccggcca cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga 360

atattgcaca atgggcgcaa gcctgatgca gcgacgccgc gtgagggatg acggccttcg 420

ggttgtaaac ctctttcagt agggaagaag cgaaagtgac ggtacctgca gaagaagcgc 480

cggctaacta cgtgccagca gccgcggtaa tacgtagggc gcaagcgtta tccggaatta 540

ttgggcgtaa agagctcgta ggcggtttgt cgcgtctgct gtgaaagacc ggggctcaac 600

tccggttctg cagtgggtac gggcagacta gagtgcagta ggggagactg gaattcctgg 660

tgtagcggtg aaatgcgcag atatcaggag gaacaccgat ggcgaaggca ggtctctggg 720

ctgtaactga cgctgaggag cgaaagcatg gggagcgaac aggattagat accctggtag 780

tccatgccgt aaacgttggg cactaggtgt gggggacatt ccacgttttc cgcgccgtag 840

ctaacgcatt aagtgccccg cctggggagt acggccgcaa ggctaaaact caaaggaatt 900

gacgggggcc cgcacaagcg gcggagcatg cggattaatt cgatgcaacg cgaagaacct 960

taccaaggct tgacatgaac cggaaagacc tggaaacagg tgccccgctt gcggtcggtt 1020

tacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080

cgagcgcaac cctcgttcta tgttgccagc gcgttatggc ggggactcat aggagactgc 1140

cggggtcaac tcggaggaag gtggggacga cgtcaaatca tcatgcccct tatgtcttgg 1200

gcttcacgca tgctacaatg gccggtacaa agggttgcga tactgtgagg tggagctaat 1260

cccaaaaagc cggtctcagt tcggattggg gtctgcaact cgaccccatg aagtcggagt 1320

cgctagtaat cgcagatcag caacgctgcg gtgaatacgt tcccgggcct tgtacacacc 1380

gcccgtcaag tcacgaaagt tggtaacacc cgaagccggt ggcctaaccc ttgtgggggg 1440

agc 1443

<210> 6

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

atgttcgcca accacgcct 19

<210> 7

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

ggataccggg cgacgttagc 20

<210> 8

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

ggactagtca tgttcgccaa ccacgcct 28

<210> 9

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

ggggtacccg gataccgggc gacgttagc 29

<210> 10

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

tgctctagag caatgttcgc caaccacgcc t 31

<210> 11

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

cgcgacgtcc gcggataccg ggcgacgtta gc 32

Claims

1. a kind of hyaluronate lyase, which is characterized in that the amino acid sequence of the hyaluronate lyase such as SEQ ID NO. shown in 1.

2. a kind of hyaluronate lyase gene, encodes hyaluronate lyase described in claim 1, which is characterized in that sequence It is classified as the complementary series of sequence shown in SEQ ID NO.2 or SEQ ID NO.2.

3. recombinant vector, it includes hyaluronate lyase genes as claimed in claim 2.

4. recombinant vector according to claim 3, which is characterized in that the transfer vector plasmid is escherichia coli plasmid PProEX-HTa or bacillus subtilis bacteria plasmid pHT43.

5. a kind of cell, which is characterized in that the cell contains hyaluronate lyase gene nucleotide as claimed in claim 2 Sequence；Or the cell containing hyaluronate lyase gene nucleotide series as claimed in claim 2 or containing having the right by wanting It seeks the transformation of host cells of 3 recombinant vectors and obtains.

6. cell according to claim 5, it is characterised in that: the host cell is Escherichia coli or bacillus subtilis Bacterium.

7. a kind of Arthrobacter globiformisArthrobacter globiformisHL6, depositary institution: Chinese Typical Representative culture is protected Hiding center, preservation date: on 07 05th, 2018, deposit number: CCTCC M 2018452.

8. a kind of method for preparing hyaluronate lyase as described in claim 1, which is characterized in that comprise the steps of:

(1) clone of hyaluronate lyase gene and analytical procedure: Arthrobacter globiformis is extractedArthrobacter globiformils2018452 genome of HL6 CCTCC M, according to genome sequence and hyaluronate lyase functional gene Analysis, design primer, using the genomic DNA of extraction as template, by PCR obtain hyaluronate lyase gene；

(2) construction step of hyaluronate lyase recombinant vector: the hyaluronate lyase gene that step (1) is obtained Nucleotide sequence is connect after double enzyme digestions with escherichia coli plasmid pProEX-HTa, obtains Escherichia coli recombinant vector, or step Suddenly the nucleotide sequence for the hyaluronate lyase gene that (1) obtains after the digestions of pair enzyme with bacillus subtilis bacteria plasmid pHT43 Connection obtains bacillus subtilis recombinant vector；

(3) construction step of hyaluronate lyase recombinant cell: step (2) hyaluronate lyase recombinant vector is converted big Enterobacteria BL21(DE3) obtain Escherichia coli hyaluronate lyase recombinant cell, or by step (2) hyaluronate lyase weight Group carrier conversion bacillus subtilis WB800N obtains bacillus subtilis hyaluronate lyase recombinant cell；

(4) the expression and purification step of hyaluronate lyase: by the cell containing hyaluronate lyase gene or containing saturating The cell inoculation of the recombinant vector of bright matter lyase gene is cultivated into bioreactor, inducing expression, collects expression Product obtains hyaluronate lyase activated protein by affinitive layer purification, or by ion exchange with it is gel-filtration purified Obtain hyaluronate lyase activated protein.

9. a kind of composition, which is characterized in that contain hyaluronate lyase described in claim 1.

10. composition according to claim 9, which is characterized in that containing acceptable auxiliary in other food or drug Material or carrier.

11. composition according to claim 9 or 10, which is characterized in that include or not comprising pharmaceutically active agents.

12. composition according to claim 11, which is characterized in that the pharmaceutically active agents are selected from: insulin, cell The factor, antibody, chemotherapeutics, analgesic, anti-inflammatory agent, antibacterial agent, anti-amoeba worm agent, anti-Parkinson agent, anti-dysentery agent, anti-convulsion Contraction agent, antidepressant, antirheumatic, antifungal agent, rescinnamine, antipyretic, antihistaminic, α-adrenaline excitant, α Blocking agent, anesthetic, bronchodilator, biocide, beta adrenergic blocking agent, calcium channel blocking agent, cardiovascalar agent, Practise contraception medicament, decongestant agent, diuretics, sedative, diagnostic reagent, electrolyte reagent, hypnosis medicament, hormone, muscular relaxation agent, Flesh contracting agent, eye medicine, parasympathomimetics, psychic energizer, tranquillizer, sympathetic transmitter releasers, uropoiesis agent, vagina are used Any one of medicament, vitamin medicament, angiotensin converting enzyme inhibitors and rush SLEEP AGENT.

13. a kind of application of hyaluronate lyase described in claim 1, which is characterized in that be used to prepare cosmetics or change Kind good appearance.