CN110527680B

CN110527680B - Hyaluronic acid lyase, gene thereof and application thereof

Info

Publication number: CN110527680B
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: 2022-11-18
Anticipated expiration: 2039-09-06
Also published as: CN110527680A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a hyaluronic acid lyase, a gene thereof, a recombinant vector and a cell containing the gene, and expression and application of the hyaluronic acid lyase. A hyaluronan lyase, wherein the amino acid sequence of the hyaluronan lyase is shown as SEQ ID No. 1. A hyaluronic acid lyase gene codes hyaluronic acid lyase, and the sequence is a sequence shown in SEQ ID NO.2 or a complementary sequence of SEQ ID NO. 2. According to the invention, the heterologous high-efficiency active expression of the bacterial hyaluronic acid lyase is successfully realized by adopting an engineering strain (escherichia coli or bacillus subtilis) expression system, the fermentation activity is obviously higher than that of the prior report, the large-scale production of the bacterial hyaluronic acid lyase is realized, the defect that the separation and purification process of a wild strain is complex is overcome, and the high-purity hyaluronic acid lyase product is prepared, so that a foundation is laid for the industrial production of the hyaluronic acid lyase and the research and application of the hyaluronic acid lyase in the fields of biochemical engineering, medicines and the like.

Description

Hyaluronic acid lyase, gene thereof and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a hyaluronic acid lyase, a gene thereof and application thereof.

Background

Hyaluronic acid is a chain-like high-molecular polymer formed by alternatively connecting acetylglucosamine glucuronic acid, is a main component of an extracellular matrix of animal tissues, and is widely present in biological tissues such as skin, cartilage, joints, vitreous bodies and the like. The hyaluronic acid can keep water more than 500 times of the self weight to form a very viscous solution, and the solution is filled in an extracellular matrix collagen fibrous skeleton to form a physical barrier between tissues, so that a certain mass transfer resistance is caused in the process of drug delivery, and the hyaluronic acid is a main obstacle of the delivery of many drugs.

Hyaluronidase, also called hyaluronidase, is a glycosidase which can make hyaluronic acid have low molecular action, can also degrade acidic mucopolysaccharide in other body tissues, is widely present in eukaryotes and prokaryotes, is involved in many important biological processes in animal bodies, such as cell division, intercellular connection, activity of germ cells, transfection of DNA, embryonic development, and repair of injured tissues, and is an important physiologically active substance. And was first discovered and called "spreading factor" by Duran Reynals in 1928 and formally named hyaluronidase by Chain and Duthie in 1940. Hyaluronidases fall into 3 major classes: (1) The first is endo- β -N-acetylglucosaminidase (ec 3.2.1.35), which is derived mainly from the venom of vertebrates and some organisms. These enzymes are hydrolases and transglycosidases which act on the beta-1, 4 glycosidic bond to degrade the substrate to give the tetrasaccharide-based end product. (2) The second type is endo- β -glucuronidase (ec 3.2.1.36), which is mainly extracted from the salivary glands of leeches and duodenum. The enzyme is hydrolase, HAs a main action domain of beta-1, 3 glycosidic bond, can specifically degrade HA, and generates a final product mainly comprising tetrasaccharide or hexose. (3) The third type is hyaluronan lyase (EC 4.2.2.1), which is produced primarily by bacteria, acts on the beta-l, 4-glycosidic bond, and produces the 4, 5-unsaturated disaccharide by the beta-elimination mechanism.

The hyaluronidase catalyzes the hydrolysis of hyaluronic acid to reduce the viscosity of hyaluronic acid, thereby reducing the viscosity of extracellular matrix, improving the permeability of liquid in tissues, improving the tissue permeability of drugs, and promoting the diffusion and transmission of drugs. Hyaluronidase has been used for a variety of purposes such as subcutaneous infusion, periocular anesthesia, chemotherapeutics and biopharmaceutical permeation, absorption of contrast agents in urography, treatment of edema, arthritis, etc. in the medical field.

With the development of hyaluronidase, one potentially important use of hyaluronidase is: the composition is used in combination with other medicines, is used for promoting the diffusion and absorption of subcutaneous medicines, synergistically resisting tumors, improving marketed medicines and the like, is used for treating serious diseases, particularly tumors, and has great potential.

Hyaluronidase is used in combination with immunoglobulin for the treatment of primary immunodeficiency syndrome and is marketed in europe, the united states and australia, and there are currently a number of hyaluronidases in combination with immunoglobulins progressing to different clinical stages; the hyaluronidase is used in combination with insulin for treating diabetes, hereditary angioedema, hypercholesterolemia and multiple myeloma.

The deposition increase of extracellular matrix (high expression of hyaluronic acid) is a typical characteristic of a plurality of solid tumors (pancreatic cancer, prostatic cancer, breast cancer, ovarian cancer, membrane adenocarcinoma, colon cancer, gastric cancer, non-small cell lung cancer and the like), the hyaluronic acid and other components are enriched in an abnormal way to surround cancer cells to generate a protective network and support the cancer cells, and the pathological accumulation of the hyaluronic acid and other components can increase the pressure of interstitial fluid of the tumors and compress tumor blood vessels to form a unique microenvironment which is more beneficial to the growth of the cancer cells, promote the growth of the cancer cells and prevent the diffusion of antitumor drugs to the tumor cells, so that the potential efficacy of most anticancer drugs is greatly limited, and the resistance of the therapeutic drugs is caused.

In recent years, targeting of extracellular matrix, combined with specific tumor cell growth inhibitors, has become a new therapy for treating tumors. Hyaluronic acid is rapidly degraded by hyaluronidase, drug transport impedance is relieved, and then the anti-cancer therapeutic agent can be more effectively transported to a tumor target, and the treatment effect is improved. Hyaluronidase in combination with gemcitabine & paclitaxel for the treatment of pancreatic cancer; hyaluronidase is used in combination with eribulin mesylate for the treatment of metastatic breast cancer, and has been in clinical II; the hyaluronidase modification compound is combined with a PD-1 inhibitor, and is used for treating non-small cell lung cancer and the like to different clinical stages.

The hyaluronidase can also be used for improving the marketed drugs and changing the administration route of the marketed drugs. If the Herceptin (trastuzumab) and MabThera (rituximab) intravenous injection preparations are changed into subcutaneous injection preparations, the treatment time is shortened from 2 to 3 hours to 5 minutes, and the treatment time is obviously shortened. Herceptin and MabThera subcutaneous injection preparations have been approved for marketing in Europe in 2013 and 2014. HyQvia, a preparation of hyaluronidase in combination with immunoglobulin, was the first monthly subcutaneous immunoglobulin approved for the treatment of primary immunodeficiency. Only one subcutaneous injection is needed per month and the entire therapeutic dose can be delivered at a single injection site. Provides a new medication option for patients who do not want to inject and instill many times frequently. The drug product is currently marketed in the european union in 5 months 2013 and in the us in 9 months 2014.

Hyaluronic acid bulking agents can develop early and late complications ranging from mild to severe in medical cosmetics, cosmetics and skin care. Hyaluronidase can treat allergic reactions, vascular embolisms, underutilization, overuse, subcutaneous induration, delayed pigmentation, immune reactions, granuloma and other adverse reactions caused by bulking agents, and significantly improve fibrotic disorders in cellulite.

The hyaluronidase used at present is mostly derived from animal tissues and belongs to hyaluronidase. Several animal-derived hyaluronidase products have been approved by the FDA in the united states for human use until now, but the use and production of hyaluronidase extracted from animal tissues is greatly limited due to its low purity, high impurity content, high sensitivity, high immunogenicity, and potential risk of transmitting spongiform encephalopathy in animals. Wherein Hyase and Wydase have been released from the market, vitrase (sheep testis extract) and Amphadase (cattle testis extract) also experience the process of releasing from the market/suspending to go into the market, the product is purified and refined to obtain high purity products, and the product is supplied with data and then continues to go into the market for circulation, and the selling price is high.

In the study of hyaluronidase recombinase, yeast, E.coli and Bacillus subtilis can be used as hosts for recombinant hyaluronidase. At present, recombinant expression of hyaluronidases such as leech hyaluronidase, human hyaluronidase, streptomyces and the like is realized, and the recombinant expression of the hyaluronidase derived from bacteria is less. The published data shows that the yield of the hyaluronidase produced by recombinant fermentation is obviously higher than that of other processes, and the purification process is simpler, the product purity is high, and the method has good biochemical and medical application prospects.

In patent CN 103614352B, a method for high efficiency recombinant expression of leech hyaluronidase is disclosed, which not only significantly improves the yield and realizes the secretory expression of leech hyaluronidase (21333.33U/mL), but also simplifies the purification steps. In patent CN 103695448B, a method for recombinant expression of hirulog hyaluronidase by pichia pastoris is disclosed, and separation and purification are performed, which provides a basis for industrial application of hirulog hyaluronidase. The patent CN 104745553B discloses a recombinant human hyaluronidase and a preparation method thereof, wherein a human hyaluronidase gene is integrated into pichia pastoris engineering bacteria, and a large amount of high-activity and high-purity recombinant human hyaluronidase is obtained through fermentation and purification. In patent CN 104342420B and patent CN 1942588B, a method for recombinant expression of soluble human hyaluronidase rHuPH20 by using Chinese Hamster Ovary (CHO) cells is disclosed, which effectively avoids the risk of spongiform encephalopathy of animals, but because the production process is complex, the cost is high, the half-life period in vivo is short, the treatment effect is influenced, and the application of hyaluronidase in medicine and industry is difficult to satisfy. A new type of adhesive is disclosed in patents CN 102439144B and CN 105473607ADerived from streptomyceskoganeiensisThe hyaluronidase and its preparation method are provided. Patent CN 103255076B discloses a hyaluronidase utilizing bacillus, a preparation method thereof and application of the hyaluronidase in preparing oligomeric hyaluronic acid or salts thereof. Sukang et al disclose a vaccine derived from Arthrobacter nicotinovorans: (A), (B), and (C)Artbrobacter nicotinovorous) The molecular weight of the hyaluronidase is 22.1kDa, but the highest enzyme production activity is only 64.32U/mL, and the enzyme activity is low, so that the hyaluronidase is not suitable for large-scale development and application. Zhang Jingliang et al in the patent "A spherical arthrobacter and its produced hyaluronidase" (application number: 201610115793.0) discloses hyaluronidase from spherical arthrobacter a152 of intertidal sludge, its molecular weight is 73.7kDa, and the highest enzyme activity of fermentation is 7100U/mL by temperature-changing regulation.

Disclosure of Invention

The applicant of the present invention provides a hyaluronan lyase, a gene thereof and an application thereof by intensive research and creative work, and also constructs a recombinant expression vector of a hyaluronan lyase gene and a host cell expressing the hyaluronan lyase. The enzyme activity of the fermentation liquor of the hyaluronic acid lyase can reach 4.9 multiplied by 10 at most ⁶ U/mL, is obviously higher than most of enzyme activities reported in the literature at present. The enzyme has good temperature stability and pH stability, high product purity, and unique advantages in preparation of low molecular glycosaminoglycan as diffusion promoter and biochemical application in medicine.

These objects, and others which will become more apparent hereinafter, are achieved according to the present invention by the following technical solution, in particular: a hyaluronidase has an amino acid sequence shown in SEQ ID NO. 1.

The nucleotide sequence of the gene for coding the hyaluronic acid lyase is shown as SEQ ID NO.2 or the complementary sequence of the SEQ ID NO. 2.

A recombinant vector comprising the above-mentioned hyaluronan lyase gene inserted into a plasmid, preferably according to the present invention, the selected plasmid is the E.coli plasmid pProEX-HTa or the Bacillus subtilis plasmid pHT43.

A cell containing the nucleotide sequence of the hyaluronan lyase gene; or the cell is obtained by transforming a host cell by a recombinant vector containing the nucleotide sequence of the hyaluronic acid lyase gene, preferably, the host cell is escherichia coli or bacillus subtilis, and more preferably, escherichia coli BL21 (DE 3) or bacillus subtilis WB800N.

A bacterial strain HL6 producing hyaluronan lyase was isolated from seawater, and the expression of the promoter in the forward primer 27F:5 '-AGAGAGTTTGATCTGCTCAG-3' (SEQ ID No: 3) and 5 'ACGGCTACCTTGTTACGACTT-3' (SEQ ID No: 4) are reversely introduced, PCR amplification is carried out to obtain a 16S rRNA sequence shown as SEQ ID No. 5, the strain 16S rRNA is 1443bp in length and has the highest similarity of 99.7 percent with Arthrobacter bacteria after the identification of 16S rRNA sequence alignment in NCBI database, and the strain is preliminarily identified as the Arthrobacter. The bacterial strain is subjected to morphological and physiological biochemical identification according to Bergey's handbook, and the result shows that the bacterial strain is cultured for 48 hours at 24 ℃, and the bacterial colony is round, colorless and transparent. Gram staining positive. Can utilize creatine, sarcosine, betaine and glycine as single carbon source, can liquefy gelatin and hydrolyze starch, but can not reduce NO ₃ To NO ₂ Biotin is required as a growth factor, ammonium sulfate and soil factor are not required as a growth factor, and leucine enzyme and glutaminase are positive, and xylose, mannose, ribose and raffinose cannot be utilized. Morphological and physiological and biochemical results show that the strain has significant difference with arthrobacter globiformis A152 reported in the invention with the application number of 201610115793.0. Combining 16 rRNA sequence analysis, morphological and physiological and biochemical identification results, the strain is named as arthrobacter globiformis (R) ((R))Arthrobacter globiformis）HL6。

Arthrobacter globiformis (A), (B) and (C)Arthrobacter globiformis) HL6 is preserved in the China center for type culture Collection, and the preservation date is as follows: year 2018, month 07, day 05, accession number: CCTCC M2018452.

A method for preparing the hyaluronan lyase, characterized by comprising the steps of:

1) Hyaluronic acid lyase baseCloning and analysis of causes: extraction of Arthrobacter globiformis (B)Arthrobacter globiformis) Designing a primer according to the analysis of a genome sequence and a functional gene of the hyaluronic acid lyase by using an HL6 CCTCC M2018452 genome, and obtaining a hyaluronic acid lyase gene by PCR (polymerase chain reaction) by using extracted genome DNA as a template;

2) Construction of recombinant vector for hyaluronan lyase: carrying out double-enzyme digestion on the obtained nucleotide sequence of the hyaluronic acid lyase gene, and then connecting the obtained nucleotide sequence with an escherichia coli plasmid pProEX-HTa to obtain an escherichia coli recombinant vector, or carrying out double-enzyme digestion on the obtained nucleotide sequence of the hyaluronic acid lyase gene, and then connecting the obtained nucleotide sequence with a bacillus subtilis plasmid pHT43 to obtain a bacillus subtilis recombinant vector;

3) Construction of recombinant cells of hyaluronidase: transforming the recombinant vector of the hyaluronidase into escherichia coli BL21 (DE 3) to obtain recombinant cells of the hyaluronidase of the escherichia coli, or transforming the recombinant vector of the hyaluronidase into bacillus subtilis WB800N to obtain recombinant cells of the bacillus subtilis hyaluronidase;

4) Expression and purification of hyaluronan lyase: inoculating cells containing the gene of the hyaluronic acid lyase or cells of a recombinant vector containing the gene of the hyaluronic acid lyase into a bioreactor for culture, inducing expression, collecting an expression product, and purifying by affinity chromatography to obtain the active protein of the hyaluronic acid lyase, and optionally, purifying by ion exchange and gel filtration to obtain the active protein of the hyaluronic acid lyase.

A composition comprising said hyaluronan lyase. The composition further comprises other food or pharmaceutical acceptable adjuvants or carriers. Further the composition may or may not contain a pharmaceutically active agent.

Wherein the pharmaceutically active agent is selected from: any of insulin, cytokines, antibodies, chemotherapeutic agents, analgesics, anti-inflammatory agents, antibacterial agents, anti-amoeba agents, anti-parkinson agents, anti-dysentery agents, anti-spasmodics, anti-depressants, anti-rheumatic agents, antifungal agents, antihypertensive agents, antipyretics, antihistamines, alpha-adrenergic agonists, alpha blockers, anesthetics, bronchodilators, biocides, beta-adrenergic blockers, calcium channel blockers, cardiovascular agents, contraceptive agents, decongestants, diuretics, sedatives, diagnostic agents, electrolytic agents, hypnotic agents, hormones, muscle relaxants, muscle contractants, ophthalmic agents, parasympathomimetics, psychostimulants, tranquilizers, sympathomimetic agents, urologics, vaginal agents, vitamin agents, angiotensin converting enzyme inhibitors, and hypnotic agents.

The hyaluronan lyase gene or recombinant vector or recombinant cell or the arthrobacter globiformis: (Arthrobacter globiformis) Application of HL6 CCTCC M2018452 in preparing hyaluronidase is provided.

The hyaluronan lyase or the composition is used for treating and/or preventing diseases or disorders, or for cosmetic application or for improving aesthetic appearance.

Compared with the prior art, the hyaluronic acid lyase and the gene thereof have the following beneficial effects:

according to the invention, the heterologous high-efficiency active expression of the bacterial hyaluronic acid lyase is successfully realized by adopting an engineering strain (escherichia coli or bacillus subtilis) expression system, the fermentation activity is obviously higher than that of the prior report, the large-scale production of the bacterial hyaluronic acid lyase is realized, the defect that the separation and purification process of a wild strain is complex is overcome, and the high-purity hyaluronic acid lyase product is prepared, so that a foundation is laid for the industrial production of the hyaluronic acid lyase and the research and application of the hyaluronic acid lyase in the fields of biochemical engineering, medicines and the like.

Drawings

FIG. 1: arthrobacter globiformis (A), (B) and (C)Arthrobacter globiformils) An electropherogram of total DNA of HL6 CCTCC M2018452 cells in 1% agarose gel, wherein the left side is marker, and the right side is a sample;

FIG. 2 is a schematic diagram: electropherograms of the hyaluronan lyase gene in 1% agarose gels. The marker is arranged on the left side, and the sample is arranged on the right side;

FIG. 3: SDS-PAGE electrophoresis of purified hyaluronidase. The molecular weight of the standard protein is shown on the left side, and the target protein of the purified hyaluronan lyase is shown on the right side;

FIG. 4: the viscosity change of hyaluronic acid is degraded by hyaluronic acid lyase. Reaction time(s) on abscissa, and viscosity (mPa @) on ordinate;

FIG. 5: effect of temperature on hyaluronan lyase. The abscissa represents different temperatures, and the ordinate represents relative enzyme activity;

FIG. 6: the hyaluronan lyase is stable at temperatures of 4 ℃ and 37 ℃. The abscissa represents different time, and the ordinate represents relative enzyme activity;

FIG. 7: influence of pH on the hyaluronan lyase. The abscissa is different pH values, and the ordinate is relative enzyme activity;

FIG. 8: the pH stability of the hyaluronan lyase at pH 7.0. The abscissa represents different time, and the ordinate represents relative enzyme activity;

FIG. 9: effect of hyaluronidase on paclitaxel-induced apoptosis of hela cells. The horizontal axis represents the sample and concentration, and the vertical axis represents the cell viability (%).

Detailed Description

The invention realizes the recombinant expression of the hyaluronic acid lyase and prepares a high-quality hyaluronidase product with high enzyme activity and high product purity.

As used herein, the "hyaluronidase activity" is defined in units of enzyme activity. The hyaluronidase activity in the fermentation broth prepared by the above protocol was determined by the method of Chinese pharmacopoeia (refer to the general rule 1207 of Chinese pharmacopoeia 2015 edition: the hyaluronidase assay). The enzyme activity unit of the fermentation liquor is defined as: enzyme activity units (U/mL) per mL of fermentation broth.

As used herein, the "flat plate transparent ring method" operates on the following principle: hyaluronic acid is high-molecular polyanion polysaccharide, and is precipitated in a surfactant aqueous solution with a certain concentration, and hydrolyzed low-molecular hyaluronic acid cannot be precipitated. By using the principle, the activity of the hyaluronidase can be quickly and simply identified.

The hyaluronidase of the invention can be recombinant polypeptide, natural polypeptide, synthetic polypeptide. The polypeptides of the invention may be naturally purified products, or chemically synthesized products, or produced from hosts (e.g., large intestine bacteria and Bacillus subtilis) using recombinant techniques.

The invention includes fragments of a hyaluronan lyase. As used herein refers to a polypeptide that retains substantially the same biological function or activity of the native hyaluronidase of the present invention. A polypeptide fragment of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the definitions herein.

The technical scheme of the invention is further explained by combining with specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 cloning and analysis of the Hyaluronan lyase Gene

Taking Arthrobacter globiformis cultured to logarithmic phase (A)Arthrobacter globiformis) The bacterial solution of HL6 CCTCC M2018452, DNA extraction is carried out according to the operation instruction of a TIAamp Bacteria DNA Kit (TIANGEN BIOTECH (BEIJING) CO., LTD), DNA band detection is carried out in 1% agarose gel electrophoresis, and the detection conditions are as follows: the sample loading amount is 5 mu L, the voltage is 120V, electrophoresis is carried out for 30min, and a band is observed in a gel imaging system (an electrophoretogram is shown in figure 1), so that the band uniformity is good. Extracting the DNA sampleSequencing the product.

Arthrobacter globiformis (B)Arthrobacter globiformis) HL6 CCTCC M2018452 total DNA is taken as a template, functional gene analysis of a database is carried out, primers F and R are adopted to amplify a hyaluronan lyase gene HyLs:

F：ATGTTCGCCAACCACGCCT （SEQ.ID.No.6）

R：GGATACCGGGCGACGTTAGC （SEQ.ID.No.7）

PCR was used for amplification, and the 50. Mu.l reaction system was:

1 μ L of DNA template in 50 μ L of reaction system; f (10. Mu.M), 1. Mu.L; r (10. Mu.M), 1. Mu.L; dNTPs (2.5 mM each), 4. Mu.L; taq (2U/. Mu.L), 1. Mu.L; 10 × Taq buffer,5 μ L; ddH2O, to 50. Mu.L.

The PCR amplification procedure was: 95. pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 45s, annealing at 56 deg.C for 45s, extension at 72 deg.C for 1 min, and circulation at 72 deg.C for 10min for 30 times. The PCR product was analyzed by 1% agarose electrophoresis and sequenced after single specific bands (the electrophoretogram is shown in FIG. 2).

Obtaining Arthrobacter globiformis: (Arthrobacter globiformis) The expression gene HyLs of HL6 CCTCC M2018452 hyaluronic acid lyase has the length of 2298 bp, and the sequencing sequence is shown in a sequence table SEQ.ID.No. 2. The gene sequence shown in SEQ.ID.No.2 is compared with the whole genome sequence, and the two sequences are consistent. The gene codes a protein consisting of 765 amino acids, and the sequence is shown in a sequence table SEQ.ID.No. 1. Blast sequence analysis in NCBI database shows that the protein belongs to polysaccharide lyase 8 family, the similarity of the amino acid sequence and the reported hyaluronic acid lyase amino acid sequence is only up to 90.46% (Arthrobacter hyaluronic acid lyase: WP _039242887.1, WP _018778839.1), and the protein is a new protein and the gene is a new gene.

Example 2 construction of recombinant vector for Escherichia coli for Hyaluronolytic enzyme

Arthrobacter globiformis (A), (B) and (C)Arthrobacter globiformis) HL6 CCTCC M2018452 total DNA is taken as a template, and primers EC-F and EC-R containing restriction enzyme cutting sites are used for amplifying a hyaluronan lyase gene HyLs (without signal) through functional gene analysis of a databasePeptide No.:

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

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

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

PCR was used for amplification, and the 50. Mu.l reaction system was:

1 mu L of DNA template is contained in a 50 mu L reaction system; EC-F (10. Mu.M), 1. Mu.L; EC-R (10. Mu.M), 1. Mu.L; dNTPs (2.5 mM each), 4. Mu.L; taq (2U/. Mu.L), 1. Mu.L; 10 XTaq buffer, 5. Mu.L; ddH2O, to 50. Mu.L.

The PCR amplification procedure was: 95. pre-denaturation at 96 deg.C for 3 min, denaturation at 96 deg.C for 30s, annealing at 60 deg.C for 45s, extension at 70 deg.C for 90s, and circulation at 70 deg.C for 10min for 30 times.

And performing 1% agarose electrophoresis and sequencing verification on the PCR product, wherein the amplification sequence is consistent with the hyaluronic acid lyase gene HyLs shown in the sequence table SEQ.ID.No. 2. The PCR product was purified according to the protocol required for the Cycle-Pure Kit (OMEGA Bio-Tek Co.) purification Kit.

The vector pProEX-HTa and the PCR amplification gene are subjected to double enzyme digestion respectively, the digestion product is subjected to agarose electrophoresis, and the recovery is carried out according to the operation method required by a Gel extraction kit (OMEGA Bio-Tek Co.).

Connecting the fragment containing the gene sequence of the hyaluronic acid lyase gene with a vector pProEX-HTa by using T4 ligase to obtain a recombinant vector HTa-HyLs containing the hyaluronic acid lyase gene.

Example 3 expression of recombinant Hyaluronolytic enzymes in E.coli

Escherichia coli strain DH 5. Alpha. Was selected, after preparation of competent cells, heat shock transformation (42 ℃,60 s), incubation (37 ℃,160rpm, 45min), transformants were selected on LB solid plates containing 75. Mu.g/mL ampicillin sodium, and untransformed strains could not grow on the plates. PCR detection is carried out to obtain a positive clone strain, plasmid extraction is carried out by a Plasmid Mini Kit (OMEGA Bio-Tek Co.) Kit, and recombinant plasmids HTa-HyLs are obtained.

Selecting an escherichia coli expression strain BL21 (DE 3), preparing competent cells, performing heat shock transformation (42 ℃,60 s), incubating (37 ℃,45 min), transforming the extracted recombinant plasmid HTa-HyLs into the escherichia coli expression strain BL21 (DE 3), screening a 75 mu g/mL ampicillin LB solid plate, culturing at 37 ℃ for 16h to obtain transformants, selecting a single colony transformant for PCR detection, and obtaining a positive clone strain. Storing in glycerol at-80 deg.C.

The positive clone strain was inoculated into LB liquid medium (75. Mu.g/mL ampicillin sodium) and cultured at 37 ℃ to OD ₆₀₀ When the concentration is 0.6 to 0.7, adding IPTG to the final concentration of 0.25mM, inducing and culturing at 24 ℃ and 180r/min for 24h, centrifuging the fermentation liquor at 4 ℃ and 8000r/min for 10min, collecting the fermentation supernatant, and determining the activity of extracellular hyaluronidase to be 8.5 multiplied by 10 ⁶ U/mL。

Example 4 construction and expression of recombinant vector for hyaluronidase Bacillus subtilis

Arthrobacter globiformis (A), (B) and (C)Arthrobacter globiformis) HL6 CCTCC M2018452 total DNA is taken as a template, and primers BS-F and BS-R containing restriction enzyme cutting sites are used for amplifying a hyaluronidase gene HyLs (without a signal peptide) through functional gene analysis of a database:

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

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

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

PCR was used for amplification, and the 50. Mu.l reaction system was:

1 mu L of DNA template is contained in a 50 mu L reaction system; BS-F (10. Mu.M), 1. Mu.L; BS-R (10. Mu.M), 1. Mu.L; dNTPs (2.5 mM each), 4. Mu.L; taq (2U/. Mu.L), 1. Mu.L; 10 × Taq buffer,5 μ L; ddH2O, to 50. Mu.L.

Carrying out double-enzyme digestion on the vector pHT43 and the PCR amplification gene respectively, carrying out agarose electrophoresis on the digestion product, and recovering according to the operation method required by a Gel extraction kit (OMEGA Bio-Tek Co.). The fragment containing the gene sequence of the hyaluronan lyase is connected with the vector pHT43 by using T4 ligase to obtain a recombinant vector pHT43-HyLs containing the gene of the hyaluronan lyase.

Escherichia coli strain DH5 alpha was selected, by competent cell preparation, heat shock transformation (42 degrees C., 60 s), incubation (37 degrees C., 160rpm, 45min), in the presence of 75 u g/mL ampicillin sodium LB solid plate screening transformants, the untransformed strain can not grow on the plate. PCR detection is carried out to obtain a positive clone bacterial strain, and Plasmid extraction is carried out according to the method required by a Plasmid Mini Kit (OMEGA Bio-Tek Co.) Kit to obtain recombinant plasmids pHT43-HyLs. And (3) converting the extracted recombinant plasmid pHT43-HyLs into a Bacillus subtilis expression strain WB800N, screening by a 75 mu g/mL ampicillin LB solid plate, culturing at 37 ℃ for 16h to obtain a transformant, and selecting a single colony transformant for PCR detection to obtain a positive clone strain. Storing in glycerol at-80 deg.C.

The positive clone strain was inoculated into LB liquid medium (75. Mu.g/mL ampicillin sodium) and cultured at 37 ℃ to OD ₆₀₀ When the concentration is 0.6 to 0.7, adding IPTG to the final concentration of 0.25mM,24 ℃, performing induction culture at 180r/min for 24h, centrifuging the fermentation liquor at 4 ℃ at 8000r/min for 10min, collecting the fermentation supernatant, and determining the activity of the extracellular hyaluronidase to be 5.3 multiplied by 10 ⁵ U/mL, and the enzyme activity of the fermentation liquid is lower than the fermentation expression level of escherichia coli.

Example 5 Arthrobacter globiformis: (Arthrobacter globiformis) Method for producing hyaluronic acid lyase by fermenting HL6 CCTCC M2018452

The arthrobacter globiformis (A) and (B) are preparedArthrobacter globiformis) The HL6 CCTCC M2018452 strain is inoculated into a sterilized seed culture medium (the formula of the seed culture medium is as follows: 0.2 g/L sodium hyaluronate and 5 g/L glucoseg/L, peptone 2 g/L, dipotassium hydrogen phosphate 1.5 g/L, mgSO ₄ 0.5 g/L, pH 7.5), 28 ℃,100 r/min, and culturing for 12h to obtain seed liquid.

Inoculating the seed culture solution into a sterilized fermentation medium (sodium hyaluronate 2 g/L, glucose 10g/L, peptone 5 g/L, dipotassium hydrogen phosphate 1.5 g/L, mgSO) ₄ 0.5 g/L, pH 7.5), culturing at 32 deg.C and 200 r/min for 72 h to obtain fermentation liquid containing hyaluronidase, centrifuging the fermentation liquid at 4 deg.C and 8000r/min for 10min, collecting fermentation supernatant, and determining extracellular hyaluronidase activity to be 2 × 10 ⁵ U/mL, higher than most of the reported enzyme activities.

Example 6 recombinase purification

1) Performing affinity chromatography (GE) on the centrifuged fermentation supernatant in example 3 by using a nickel column, performing primary separation and purification according to a nickel column purification instruction, and collecting a pure primary separation and purification product;

2) The primarily purified product of the step 1) is reused by QFF-Sepharose ^® Purifying Fast Flow chromatographic column (GE company), balancing the column with 2 mM Tris-HCl buffer solution with pH7.5, loading, performing linear gradient elution with 20mM Tris-HCl buffer solution (pH 7.5) containing 0-1mol/L sodium chloride, detecting protein peak at 280nm, collecting components according to tubes, determining hyaluronidase activity of each tube of collected liquid, and collecting active protein fractions;

3) Further purifying the active protein fraction collected in step 2) with Sephadex G100 Gel (GE), wherein the eluent is a 20mM phosphate buffer solution with pH of 7.0, collecting according to a tube, detecting the activity of the hyaluronidase, and collecting the active protein fraction as the purified hyaluronidase (specific activity: 5.1X 10 ⁸ U/mg). The purified hyaluronidase is subjected to SDS-PAGE electrophoresis, or a single protein band (the electrophoretogram is shown in figure 3) is obtained, the molecular weight is about 80 kDa, and the result is basically consistent with the result of the sequence SEQ.ID.No.1 of the hyaluronidase presumed from the gene of the hyaluronidase in the sequence table SEQ.ID.No. 2.

Example 7 application of hyaluronic acid lyase to reducing the viscosity of hyaluronic acid

According to the method of European pharmacopoeia (EP 9.0), 1mL of 100U/mL sodium hyaluronate solution (9 g/L) and 1mL of 10g/L hyaluronic acid are mixed at 20 ℃, the continuous change of viscosity in the solution is rapidly detected under a rheometer (Rhemeter MCR 301) for 90s, a group of hyaluronic acid solution without enzyme is used as a blank control, a group of hyaluronic acid solution heated at 100 ℃ for 30 minutes and then cooled to 20 ℃ is used as a negative control, and a middle hospital hyaluronidase standard is used as a positive control. The results of the experiment are shown in fig. 4, and the results show that the samples prepared in examples 3-6 can rapidly reduce the viscosity of the hyaluronic acid in a short time, and have the same hyaluronidase activity as the hyaluronidase standard. Meanwhile, the hyaluronidase prepared by the method has potential application in the aspects of preparing low molecular hyaluronic acid, degrading extracellular matrix hyaluronic acid, promoting drug diffusion and the like.

Example 8 Effect of temperature on hyaluronic acid lyase

The hyaluronidase enzyme activity of the samples prepared in examples 3-6 is measured according to the method specified by Chinese pharmacopoeia at 20, 30, 37, 40, 50 and 60 ℃, and the relative enzyme activity is compared by a graph (the highest enzyme activity is 100%). The experimental results are shown in figure 5, and show that the hyaluronidase samples prepared in examples 3-6 have good enzyme activity within the temperature range of 30-50 ℃, and the optimal reaction temperature is 37 ℃.

The samples of examples 3 to 6 were stored at 4 ℃ and 37 ℃ for 6 hours, sampled every 1 hour, and the enzyme activities of the samples were measured according to the enzyme activity measuring method specified in the Chinese pharmacopoeia to examine the temperature stability of the samples and compare the relative enzyme activities (the highest enzyme activity was 100%). The experimental results are shown in figure 6, and show that the hyaluronidase samples prepared in examples 3-6 are stable at 4 ℃, have small change of enzyme activity after 6 hours of storage, still contain more than 95% of the initial enzyme activity, and tend to be stable. The enzyme activity of each sample is greatly reduced by about 10% in 1 hour at 37 ℃, and then the enzyme activity tends to be stable and slowly reduced, more than 80% of the initial enzyme activity is still kept after the sample is stored for 6 hours at 37 ℃, and the temperature stability is good.

Example 9 Effect of pH on hyaluronic acid lyase

Preparing a hyaluronic acid substrate by using purified water with pH of 3-10 according to the requirements of pharmacopoeia, measuring the enzyme activity of the hyaluronidase of the samples prepared in the examples 3-6 under different pH values, and comparing the relative enzyme activity (the highest enzyme activity is 100%). The experimental results are shown in figure 7, and show that the optimal pH values of the hyaluronidase samples prepared in examples 3-6 are all 7.0, the hyaluronidase samples have good enzyme activity (the relative enzyme activity is more than or equal to 80%) in the range of pH6-8, and the enzyme activity of the hyaluronidase can be completely inhibited by higher pH (more than or equal to pH 10) and lower pH (less than or equal to pH 4).

The samples prepared in examples 3 to 6 were stored at pH7.0 (room temperature) for 6 hours, sampled every 1 hour, and the enzyme activities of the samples were measured according to the enzyme activity measuring method specified in the chinese pharmacopoeia to examine the pH stability of the samples and compare the relative enzyme activities (the highest enzyme activity was 100%). The experimental results are shown in fig. 8, and the experimental results show that the hyaluronidase samples prepared in examples 3-6 have relatively more enzyme activity decrease at 1 hour and about 10% decrease at the optimum pH of 7.0 (room temperature), and then can maintain relatively stable and slowly decrease, and still have more than 80% of relative enzyme activity at 6 hours and are relatively stable, so that the hyaluronidase samples prepared by the scheme have good pH stability. The temperature stability data of the embodiment 8 show that the prepared hyaluronan lyase sample, whether being recombinant fermentation of escherichia coli, recombinant fermentation of bacillus subtilis, fermentation of wild strains and recombinant fermentation of purified enzyme products, has high yield, high enzyme activity, stable property, the same optimal temperature and pH, good temperature and pH stability, and good development and application prospects in the aspects of biomedicine, biochemical engineering and the like.

Example 10 Effect of Hyaluronolytic enzyme on paclitaxel-induced apoptosis of human cervical carcinoma cells (Hela cells)

Hela cells were cultured at 1X 10 ⁴ Inoculating 96-well plate in each well, adding the drug to be detected (paclitaxel) after the cells adhere to the wall&Hyaluronan lyase (prepared by purification in example 6)). In DMEM medium containing 10% calf serum, 100U/mL penicillin and 100ug/mL streptomycin at 37 deg.C、5% CO ₂ The culture was carried out for 48 hours under saturated humidity. After completion of the culture, the absorbance of each well was measured at a measurement wavelength of 570nm by the MTT method. Calculating the influence of the drug on cell proliferation according to the absorbance of each hole, and calculating a cell survival rate formula: cell survival (%) = drug-containing well OD average/control well OD average 100, inhibition = 1-survival. The experimental result is shown in figure 9, and the experimental result shows that when the concentration of paclitaxel is 0.01uM, the concentration has no inhibition effect on the growth of cells compared with the blank control group, and when 1U/mL,10U/mL and 100U/mL of hyaluronidase is added respectively, the growth of cells is also not inhibited, which indicates that the hyaluronidase itself has no inhibition effect on the growth of hela cells. When the concentration of paclitaxel is 0.1uM, paclitaxel has obvious inhibition effect on the growth of hela cells, the survival rate of hela cells is about 76.5%, after hyaluronic acid lyase with different concentrations is added, the inhibition effect of paclitaxel on hela cells is gradually enhanced along with the increase of the addition amount, the survival rate is obviously reduced, when 10U/mL is added, the survival rate of hela cells is about 65.5%, when the concentration is continuously increased to 100U/mL, the survival rate is about 61.2%, the inhibition rate of paclitaxel and hyaluronic acid lyase on hela cells is increased from 23.5% to 38.8%, and the inhibition effect of paclitaxel on the growth of hela cells is obviously promoted. The reason may be that the hyaluronidase degrades high concentration hyaluronic acid in the microenvironment of tumor cells, so that paclitaxel can reach the surface of tumor cells more easily, and then the drug effect is exerted.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for some of the features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding claims.

Sequence listing

<110> Qingdao Marine Biomedicine institute, inc

<120> hyaluronic acid lyase, gene and application thereof

<140> 201910842217X

<141> 2019-09-06

<150> 2018113109388

<151> 2018-11-06

<160> 11

<170> SIPOSequenceListing 1.0

<210> 11

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<212> PRT

<213> Arthrobacter globiformis

<400> 11

Met Phe Ala Asn His Ala Trp Ala Asp Ala Glu Pro Gly Thr Ala Glu

1 5 10 15

Phe Ala Ala Leu Arg Ser Arg Trp Val Asp Gln Ile Thr Gly Arg Asn

20 25 30

Val Ile Gln Ala Gly Asp Pro Asp Phe Ala Arg Ala Val Thr Ala Leu

35 40 45

Asn Thr Lys Ala Ala Asp Ser Leu Ala Lys Leu Asn Arg Val Ser Gly

50 55 60

Arg Thr Ser Val Phe Thr Asp Leu Ser Phe Ala Lys Asp Ala Glu Met

65 70 75 80

Val Thr Thr Tyr Thr Arg Leu Ser Gln Leu Ala Ala Ala Trp Ala Thr

85 90 95

Pro Thr Ala Ala Val Phe Gly Asp Ser Ala Val Leu Ala Asp Ile Lys

100 105 110

Ala Gly Leu Ala Asp Ala Asn Thr Leu Cys Tyr His Ala Gly Arg Glu

115 120 125

Glu Val Gly Asn Trp Trp Ser Trp Glu Ile Gly Val Pro Arg Ala Leu

130 135 140

Ala Asp Ala Met Val Leu Leu His Ala Glu Leu Ser Ala Ala Glu Ile

145 150 155 160

Gln Ala Tyr Ser Ala Ala Ile Asp His Phe Val Pro Asp Pro Trp Leu

165 170 175

Gln Phe Pro Pro Lys Arg Gly Lys Ile Thr Ser Val Gly Ala Asn Arg

180 185 190

Val Asp Leu Cys Gln Gly Val Ile Ile Arg Ser Leu Ala Gly Glu Asp

195 200 205

Pro Gly Lys Leu Asn His Ala Val Ala Gly Leu Ser Gln Val Trp Gln

210 215 220

Tyr Val Thr Ser Gly Asp Gly Ile Phe Arg Asp Gly Ser Phe Ile Gln

225 230 235 240

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

245 250 255

Gly Leu Ser Lys Leu Phe Ser Leu Leu Gly Gly Thr Ala Phe Glu Val

260 265 270

Ser Asp Pro Ser Arg Ser Ile Phe Phe Asp Ala Val Glu Gly Ser Phe

275 280 285

Ala Pro Val Met Ile Asn Gly Ala Met Ala Asp Ser Val Arg Gly Arg

290 295 300

Ser Ile Ser Arg Glu Ala Asn Thr Gly Tyr Asp Leu Gly Ala Ser Ala

305 310 315 320

Ile Glu Ala Ile Leu Leu Leu Ala Arg Ala Met Asp Pro Ala Thr Ala

325 330 335

Thr Arg Trp Arg Gly Leu Cys Ala Gly Trp Ile Ala Arg Asn Thr Tyr

340 345 350

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

355 360 365

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

370 375 380

Arg Leu Phe Pro Ala Met Asp Arg Thr Met His Arg Gly Pro Gly Trp

385 390 395 400

Ala Leu Ser Leu Ser Leu Ser Ser Asn Arg Ile Ala Trp Tyr Glu Cys

405 410 415

Gly Asn Gly Glu Asn Asn Arg Gly Tyr His Thr Gly Ser Gly Met Thr

420 425 430

Tyr Phe Tyr Thr Ser Asp Leu Gly Gln Tyr Asp Asp Ala Phe Trp Ala

435 440 445

Thr Ala Asn Tyr Asn Arg Leu Pro Gly Ile Thr Val Asp Thr Thr Pro

450 455 460

Leu Pro Asp Lys Val Glu Gly Glu Trp Gly Ala Ala Val Pro Ala Asn

465 470 475 480

Glu Trp Ser Gly Ala Thr Ala Leu Gly Gly Val Ala Ala Val Gly Gln

485 490 495

His Leu Val Gly Pro Gly Arg Thr Gly Leu Ser Ala Arg Lys Ser Trp

500 505 510

Phe Val Ser Gly Glu Ala Thr Val Cys Leu Gly Ala Asp Ile Thr Thr

515 520 525

Gly Ser Gly Ala Arg Val Glu Ser Ile Val Asp His Arg Asn Leu His

530 535 540

Gln Gly Ser Asn Thr Leu Thr Thr Ala Ala Gly Thr Ile Ala Gly Ser

545 550 555 560

Val Gly Ser Ala Glu Val Leu Ser Glu Glu Arg Trp Val His Leu Glu

565 570 575

Gly Phe Gly Gly Tyr Ala Met Leu Asp Asp Ser Pro Leu His Val Leu

580 585 590

Arg Glu Thr Arg Ser Gly Ser Trp Ser Gly Val Asn Thr Asn Gly Ser

595 600 605

Thr Thr Val His Gln Arg Thr Phe Ala Thr Leu Tyr Val Asp His Gly

610 615 620

Ala Gly Pro Ala Ala Gly Ser Tyr Ala Tyr Val Val Ala Pro Gly Ala

625 630 635 640

Ser Val Asn Leu Thr Arg Lys Leu Val Gln Gly Asp Lys Tyr Arg Val

645 650 655

Ile Arg Asn Asp Thr Thr Ala Gln Ser Val Glu Phe Lys Ala Ser Lys

660 665 670

Thr Thr Ala Ala Thr Phe Trp Lys Pro Gly Met Ala Gly Asp Leu Gly

675 680 685

Ala Ser Gly Pro Ala Cys Val Val Phe Ser Arg His Gly Asn Glu Leu

690 695 700

Ser Leu Ala Phe Ser Glu Pro Thr Gln Lys Ala Ala Ser Leu Thr Leu

705 710 715 720

Thr Leu Pro Gln Gly Thr Trp Ser Ser Val Leu Glu Gly Thr Gly Thr

725 730 735

Leu Gly Thr Asp Ala Asp Gly Arg Ser Thr Val Thr Leu Asp Thr Ala

740 745 750

Gly Leu Asn Gly Gln Thr Lys Val Ile Thr Leu Arg Arg

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 hyaluronan lyase, characterized in that the amino acid sequence of the hyaluronan lyase is represented by SEQ ID No. 1.

2. A hyaluronan lyase gene encoding the hyaluronan lyase of claim 1 having the sequence shown in SEQ ID No.2 or the complement of SEQ ID No. 2.

3. A recombinant vector comprising the hyaluronan lyase gene of claim 2.

4. The recombinant vector according to claim 3, wherein the recombinant vector plasmid is the E.coli plasmid pProEX-HTa or the Bacillus subtilis plasmid pHT43.

5. A cell comprising the nucleotide sequence of the hyaluronan lyase gene of claim 2; or the cell is transformed with a host cell containing the nucleotide sequence of the hyaluronan lyase gene of claim 2 or the recombinant vector of claim 3.

6. The cell of claim 5, wherein: the host cell is escherichia coli or bacillus subtilis.

7. Arthrobacterium globiformisArthrobacter globiformisHL6, depository thereof: china center for type culture Collection, preservation date: year 2018, month 07, day 05, accession number: CCTCC M2018452.

8. A method for preparing the hyaluronan lyase of claim 1, comprising the steps of:

(1) Cloning and analyzing the hyaluronic acid lyase gene: extraction of Arthrobacter globiformisArthrobacter globiformilsAn HL6 CCTCC M2018452 genome, designing a primer according to the analysis of a genome sequence and a functional gene of the hyaluronan lyase, and obtaining the gene of the hyaluronan lyase by PCR by taking the extracted genome DNA as a template;

(2) The construction steps of the recombinant vector of the hyaluronan lyase are as follows: carrying out double-enzyme digestion on the nucleotide sequence of the hyaluronic acid lyase gene obtained in the step (1) and then connecting the nucleotide sequence with an escherichia coli plasmid pProEX-HTa to obtain an escherichia coli recombinant vector, or carrying out double-enzyme digestion on the nucleotide sequence of the hyaluronic acid lyase gene obtained in the step (1) and then connecting the nucleotide sequence with a bacillus subtilis plasmid pHT43 to obtain a bacillus subtilis recombinant vector;

(3) The construction steps of the hyaluronic acid lyase recombinant cell are as follows: converting the hyaluronidase recombinant vector in the step (2) into escherichia coli BL21 (DE 3) to obtain escherichia coli hyaluronidase recombinant cells, or converting the hyaluronidase recombinant vector in the step (2) into bacillus subtilis WB800N to obtain bacillus subtilis hyaluronidase recombinant cells;

(4) Expression and purification steps of hyaluronan lyase: inoculating cells containing the gene of the hyaluronic acid lyase or cells of a recombinant vector containing the gene of the hyaluronic acid lyase into a bioreactor for culture, inducing expression, collecting an expression product, and purifying by affinity chromatography to obtain the active protein of the hyaluronic acid lyase, or purifying by ion exchange and gel filtration to obtain the active protein of the hyaluronic acid lyase.

9. A composition comprising the hyaluronan lyase of claim 1.

10. The composition of claim 9, comprising an adjuvant or carrier acceptable for other food or pharmaceutical products.

11. The composition of claim 9 or 10, comprising or not comprising a pharmaceutically active agent.