A heparanase and its preparation method and application

Abstract

The application belongs to the technical field of bioengineering technology, and particularly relates to a heparanase and a preparation method and application thereof. The amino acid sequence of the heparanase (HPSE) is shown as SEQ ID NO. 2. The heparanase (WJS HPSE) provided in the application can specifically degrade HS glycan substrates, which are white jade snail polysaccharide (WJS PS) containing IdoA2S-GlcNAc disaccharide repeating units, heparin or HS. In addition, the heparanase (WJS HPSE) provided in the application has high thermal stability, has optimal enzyme activity at 57-67 DEG C and pH=4.0, significantly improves the stability and reuse efficiency in a catalytic process, and thus improves the application value of the HPSE.

Description

Technical Field

[0001] This application belongs to the field of bioengineering technology, specifically relating to a heparan sulfate enzyme, its preparation method, and its application. Background Technology

[0002] Heparan sulfate (HS) is composed of repeated... N Linear sulfated glycosaminoglycans are composed of disaccharide units of acetylglucosamine (GlcNAc) and D-glucuronic acid (GlcA) or L-iduronic acid (IdoA). Precise modification during HS biosynthesis—including… N - Sulfation (GlcNAc→GlcNS), C5 epimerization (GlcA→IdoA) and O -Sulfation (2- O / 6- O / 3- O (Position) – By altering the charge distribution, conformational plasticity, and protein-specific binding sites of glycans, it regulates key biological processes such as cell adhesion, signal transduction, coagulation, and viral invasion. N - Sulfation is the initiating step of HS modification, determining subsequent C5 epimerization and O -Sulphation site distribution. C5 epimerization generates more flexible IdoA through conformational inversion, enhancing the binding specificity and affinity of HS to proteins. O - Sulfation introduces a sulfate group at a specific position in IdoA or GlcNAc, forming a specific binding site for protein interaction.

[0003] The clinical anticoagulant heparin shares the same biosynthetic pathway as HS, and its glycan backbone structure is identical (the disaccharide repeating unit is a uronic acid linked to glucosamine via a 1,4 glycosidic bond). However, their modification patterns differ significantly. In heparin's typical disaccharide unit, GlcA is predominantly in its epimeric form, IdoA (accounting for approximately 90%), and its 2-position is frequently sulfated (IdoA2S). Glucosamine, on the other hand, is almost entirely... N - The disaccharide is modified with sulfated form (GlcNS, >90%), and 6-O sulfated form (GlcNS6S) is frequently observed, with some also exhibiting 3-O sulfated form (GlcNS3S6S), showing an overall extremely high sulfation density (2-3 sulfate groups per disaccharide). In contrast, the HS structure is predominantly a GlcA-GlcNAc disaccharide structure, with highly regional heterogeneous sugar chain modifications. In the low-sulfation region, uronic acid is mostly unsulfated GlcA, and glucosamine only undergoes 6-O sulfated form (GlcA-GlcNS); although the high-sulfation region is similar to the heparin structure (e.g., GlcA / IdoA2S-GlcNS6S), the proportion of IdoA is less than 50%.N - The sulfation rate is only 10%-50%, and the overall sulfation density is about one sulfate group per disaccharide. This difference makes heparin a potent anticoagulant due to its high sulfation and structural homogeneity, while HS achieves diverse biological functions such as cell signaling through heterogeneous modification of its segments.

[0004] Human heparanase (hHPSE) is the only endoglucosidase in mammals that specifically cleaves the β-1,4 glycosidic bonds in the heparin chain of heparinidase (HS). The maturation of hHPSE involves complex post-translational processing and modification. Its biosynthesis first generates a 65 kDa precursor protein (proHPSE), which is cleaved by cathepsin L to form an 8 kDa small subunit (Q36-E109) ​​and a 50 kDa large subunit (K159-I543). These two subunits are covalently linked by disulfide bonds (Cys127-Cys179 and Cys437-Cys542) to form an active dimer, participating in the regulation of HS metabolic remodeling and playing a crucial role in pathological processes such as tumor metastasis, angiogenesis, and inflammatory responses. Currently, hHPSE is widely used in antitumor drug development, low molecular weight heparin preparation, and glycan structure analysis.

[0005] The maturation mechanism of hHPSE protein is a major limiting factor for its recombinant expression. While mammalian expression systems (such as CHO cells) can complete glycosylation modification and precursor protein cleavage, the product contains uncleaved proHPSE, leading to complex purification processes and low yields. In prokaryotic or insect cell expression systems, the lack of endogenous protease cleavage capacity necessitates the construction of separate expression plasmids for each subunit, followed by co-transfection to assemble the mature enzyme. This significantly increases the difficulty of plasmid construction, virus packaging, and purification, severely restricting its industrial application prospects. Furthermore, the optimal enzyme activity temperature for the natural form of hHPSE is 37°C, exhibiting poor thermal stability and making it unsuitable for the high-temperature reaction conditions required for industrial production.

[0006] hHPSE substrate specificity is strictly dependent on N - Sulfated modification (GlcA-GlcNS disaccharide structure) lacks the ability to cleave low-sulfated HS sugar chains (such as the unmodified GlcNAc region). Unlike hHPSE, a novel bacterial heparinase (derived from...) has been discovered in recent years... Burkholderia pseudomalleiIt can specifically cleave the glycosidic bond between GlcA-linked low-sulfate domains (GlcA-GlcNAc / GlcNAc6S / GlcNS), but has no cleavage activity for the glycosidic bond between IdoA-linked low-sulfate domains (IdoA / IdoA2S-GlcNAc / GlcNAc6S / GlcNS) or GlcA-linked high-sulfate regions (GlcA-GlcNS6S / GlcNS6S3S). Furthermore, existing heparin cleavage enzymes (such as heparinase I, II, and III) can cleave both the 1,4 glycosidic bond between GlcA / IdoA with low-sulfate regions and the 1,4 glycosidic bond between IdoA2S / GlcA2S with high-sulfate regions, but their β-elimination catalytic mechanism leads to the loss of C5 chiral information in the uronic acid.

[0007] In summary, the thermostability of currently discovered heparin lyases is insufficient to meet industrial requirements; existing heparan sulfate enzymes can only cleave a limited range of substrate structures, and the resolution of highly sulfated and low-sulfated regions depends on different enzymes, limiting the precise resolution of complex glycan structures (such as heparin or HS). Therefore, exploring novel heparan sulfate enzyme resources with substrate specificity and thermostability, and constructing a complementary heparin cleavage tool enzyme library, will not only facilitate the precise structural resolution of heparin, HS, and HS structural analogs, but also provide a structural basis for the targeted preparation of specific pharmacological oligosaccharide fragments and the rational design of heparinase family inhibitors. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a heparan sulfate enzyme, its preparation method, and its application. Specifically, the following technical solution is adopted:

[0009] In a first aspect, the present invention provides a heparan sulfate enzyme, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] SEQ ID NO.2:

[0011] *

[0012] This invention discloses a recombinant preparation method, enzymatic characterization, and biomedical applications of a novel heparanase (WJS HPSE) derived from the white jade snail (WJS). The method involves isolating the WJS HPSE coding sequence from WJS using gene cloning technology, achieving recombinant expression using an insect baculovirus expression system, and obtaining a high-purity active enzyme through affinity chromatography. WJS HPSE exhibits optimal catalytic activity at pH 4.0 and 57℃, efficiently and specifically cleaving the β-1,4 glycosidic bond between IdoA2S or GlcA and GlcNAc (such as its endogenous substrate, white jade snail polysaccharide). WJS HPSE demonstrates significantly better thermal stability than human HPSE (hHPSE) in Cu-containing environments. 2+ Zn 2+ The activity is significantly enhanced in systems containing phosphate anions. These properties provide a new strategy for developing targeted degradation tools for HS or heparin glycans. The WJS HPSE recombinant preparation and enzymatic functional analysis system established in this invention provides an innovative solution for applications such as HS glycan structure analysis and low molecular weight heparin preparation.

[0013] Secondly, the present invention provides a gene encoding the above-mentioned heparan sulfate enzyme, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0014] SEQ ID NO.1:

[0015]

[0016] Thirdly, the present invention provides a recombinant expression vector comprising the aforementioned genes.

[0017] As a further preferred embodiment, the recombinant expression vector is obtained by cloning the above-described gene into the expression vector pFastBac1.

[0018] Fourthly, the present invention provides a recombinant strain comprising the above-mentioned gene or the above-mentioned recombinant expression vector.

[0019] Fifthly, the present invention provides a method for preparing heparan sulfate enzyme, characterized by comprising the following steps:

[0020] The recombinant expression vector described above was used to transform DH10Bac competent cells, and the cells were purified to obtain the recombinant plasmid.

[0021] The recombinant plasmid was transfected into SF9 cells and placed in cell culture medium for protein expression. Then, the protein was purified using affinity chromatography to obtain the heparan sulfate enzyme.

[0022] In a sixth aspect, the present invention provides the application of the above-mentioned heparan sulfate enzyme in the preparation of low molecular weight heparin.

[0023] The preparation method of this low molecular weight heparin is as follows: the above WJS HPSE recombinase is added to the reaction system containing heparin, and the low molecular weight heparin fragment is obtained after lysis.

[0024] In a seventh aspect, the present invention provides the application of the above-mentioned heparan sulfate enzyme in the preparation of low molecular weight HS.

[0025] The preparation method of the low molecular weight HS is as follows: the above WJS HPSE recombinase is added to the reaction system containing HS, and the low molecular weight HS fragment is obtained after cleavage.

[0026] Eighthly, the present invention provides the application of the above-mentioned heparan sulfate enzyme in glycan desorption.

[0027] In a ninth aspect, the present invention provides a method for reducing the glycan chains of endogenous heparan sulfate in cells, the method comprising the following steps:

[0028] The above-mentioned genes were cloned into the pLVX-IRES-Puro vector, and the ligation product was transformed into DH5α competent cells to construct the pLVX-IRES-Puro-WJS HPSE lentiviral expression vector.

[0029] The pLVX-IRES-Puro-WJS HPSE lentiviral expression vector was co-transfected with psPAX2 plasmid and pMD2.G plasmid into HEK293T cells to obtain lentivirus. HEK293 cells were then infected with lentivirus and cultured to obtain stable HEK293 cells expressing WJS HPSE. WJS HPSE Cell line.

[0030] The beneficial effects of this invention are as follows:

[0031] (1) The WJS HPSE-specifically degradable HS-type sugar chain substrate provided by the present invention is white snail polysaccharide (WJS PS, extraction method referred to patent CN114478816A) containing IdoA2S-GlcNAc disaccharide repeating unit, heparin or HS. The essential structural feature of the WJSHPSE substrate is GlcA / IdoA2S-GlcNAc.

[0032] (2) The WJS HPSE provided by the present invention has high thermal stability and has the best enzyme activity at 57 ℃-67 ℃ and pH=4.0, which significantly improves its stability and reusability in catalytic processes and has extremely high engineering application value.

[0033] (3) This invention achieves efficient recombinant preparation and enzyme catalytic characterization of WJS HPSE for the first time through genetic engineering and enzyme characteristic analysis, providing a new tool enzyme for HS metabolism research and HS glycan structure analogue analysis, and laying a theoretical and technical foundation for the development of non-mammal heat-stable heparan sulfate enzyme and the expansion of glycan-directed degradation technology industrial applications. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 The image shown is a polyacrylamide gel electrophoresis pattern (Coomassie brilliant blue staining) of WJS HPSE recombinase.

[0036] Figure 2The figures show the cleavage efficiency of WJS HPSE for different substrates; A represents the TBE-PAGE patterns (Alcian blue-silver nitrate double staining) of WJS HPSE cleavage of WJS PS polysaccharide (composed of IdoA2S-GlcNAc disaccharide repeating units), K5 polysaccharide (composed of GlcA-GlcNAc disaccharide repeating units), K5-NS polysaccharide (composed of GlcA-GlcNS disaccharide repeating units), Epi-K5 polysaccharide (composed of IdoA-GlcNS disaccharide repeating units), heparin, and HS; B represents the TBE-PAGE patterns (Alcian blue-silver nitrate double staining) of human HPSE (hHPSE) cleavage of WJS PS polysaccharide, K5 polysaccharide, K5-NS polysaccharide, Epi-K5 polysaccharide, heparin, and HS.

[0037] Figure 3 The effects of temperature and pH on WJS HPSE enzyme activity are shown (TBE-PAGE electrophoresis and Alcian blue-silver nitrate double staining analysis); A shows the effect of different temperatures on the WJS HPSE enzyme activity against WJS PS substrate; B shows the effect of different pH values ​​on the WJS HPSE enzyme activity against WJS PS substrate.

[0038] Figure 4 The effect of the enzyme digestion buffer system on the activity of WJS HPSE enzyme (TBE-PAGE electrophoresis and Alcian blue-silver nitrate double staining analysis) is shown in Figure A, where A represents the effect of metal ions on the activity of WJS HPSE enzyme digesting WJS PS substrate; and B represents the effect of anions on the activity of WJS HPSE enzyme digesting WJS PS substrate.

[0039] Figure 5 The image shows the enzymatic cleavage effect of WJS HPSE on endogenous HS glycans in cells; A represents the effect of Western blot analysis, showing that it is similar to wild-type HEK293 cells (HEK293...). 对照 Compared to HEK293, WJS HPSE WJS HPSE is expressed in cells; the B in it is related to HEK293. 对照 Compared to other cells, HEK293 cells expressing WJS HPSE... WJS HPSE Endogenous HS glycans in cells were significantly degraded (TBE-PAGE electrophoresis and Alcian blue-silver nitrate double staining analysis). Detailed Implementation

[0040] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0041] Example 1

[0042] Obtain the WJS HPSE coding gene sequence

[0043] Total RNA was extracted from *Solanum lyratum*, and cDNA was synthesized via reverse transcription using the SMARTer PCR cDNA Synthesis Kit (Clontech). Full-length transcriptome sequencing was performed using the PacBio third-generation sequencing platform, and gene function annotation was completed through bioinformatics analysis. The candidate gene sequence WJS HPSE was obtained through screening. Specific segmented primers were designed based on this sequence (primer sequences are shown in Table 1 below), and multi-fragment PCR amplification was performed on the *Solanum lyratum* cDNA. After sequencing verification of the amplified products, the full-length coding sequence of the WJS HPSE gene was obtained through sequence assembly.

[0044] Table 1 Segmented Primer Sequences

[0045]

[0046] Example 2

[0047] Recombinant expression of WJS HPSE

[0048] use QuickCut EcoR I and QuickCut Xho I Enzymes (Takara Bio Engineering (Dalian) Co., Ltd.) contain... EcoR I and Xho I The WJS HPSE sequence (synthesized by Qingke Biotechnology Co., Ltd.) and the pFastBac1 vector were double-digested (reacted at 37 ℃ for 30 minutes). The reaction system is as follows:

[0049] Table 2. Double enzyme digestion reaction system

[0050]

[0051] After separation and purification by agarose gel electrophoresis, the enzyme digestion products were ligated into the linearized pFastBac1 vector using T4 DNA ligase. The ligation product was transformed into DH5α competent cells and plated on Luria-Bertani (LB) solid medium containing ampicillin (100 μg / mL). After overnight incubation at 37 °C, single colonies were picked and inoculated into 5 mL of LB liquid medium containing ampicillin resistance, and cultured at 37 °C with shaking until the OD600 nm value reached 0.6-0.8. 5 µL of this bacterial culture was then transferred to 50 mL of LB liquid medium containing ampicillin resistance and cultured at 37 °C with shaking for 12-16 hours. Plasmids were extracted using the Endo-Free Plasmid Mini Kit II (Omega Bio-Tek). The recombinant plasmid was digested using the same double enzyme digestion system described above. Agarose gel electrophoresis showed the expected size of the fragment (WJS HPSE insert and pFastBac1 vector backbone), preliminarily confirming successful construction. Further DNA sequencing (Qingke Biotechnology Co., Ltd.) confirmed the sequence was correct, and the recombinant plasmid pFastBac1-WJS HPSE was finally obtained.

[0052] The pFastBac1-WJS HPSE recombinant plasmid was transformed into DH10Bac competent cells. After incubation at 37 °C with shaking for 48 hours, the bacterial culture was plated onto LB blue-white screening medium containing 7 µg / mL gentamicin, 50 µg / mL kanamycin, and 10 µg / mL tetracycline, with a surface coating of 2% 5-bromo-4-chloro-3-indole-β-D-galactoside (X-Gal) and 20% isopropyl-β-D-1-thiogalactoside (IPTG). The medium was incubated at 37 °C for 48 hours. The recombinant baculovirus plasmid was identified using the blue-white screening method. White single colonies were picked and inoculated into 500 µL of LB liquid medium containing kanamycin, gentamicin, and tetracycline. After amplification incubation at 37 °C with shaking for 4 hours... The bacterial culture was diluted 100-fold with antibiotic-free LB medium. 50 µL of the diluted culture was evenly spread onto the LB blue-white screening solid medium and incubated at 37 °C for 48 hours. If only white colonies were observed, it indicated that the selected white colonies contained the successfully constructed recombinant baculovirus plasmid. The recombinant baculovirus plasmid was then extracted from the amplified bacterial culture using the Endo-Free Plasmid Mini Kit II (Omega Bio-Tek). PCR amplification of the recombinant baculovirus plasmid was performed using the PUC / M13 forward and reverse primers flanking the mini attTn7 site (primer sequences: pUC / M13 F: 5'-CCCAGTCACGACGTTGTAAAACG-3'; pUC / M13 R: 5'-AGCGGATAACAATTTCACACAGG-3'). Sequencing of the PCR product confirmed the successful construction of the recombinant baculovirus plasmid, which was named Bacmid-WJS HPSE.

[0053] SF9 insect cells were loaded at 4×10 5 Cells were seeded at a density of 500 μL / mL in 24-well plates and cultured at 27°C until the cell density reached 80%. Serum-free medium containing SF-900Ⅱ, different doses of Bacmid-hHPSE recombinant baculovirus plasmid, and TransIT were then added. ®The DNA-transfection complex of the Insect Transfection reagent was used. After culturing at 27 °C for 72 hours, the cell culture supernatant was collected, and the expression level of WJS HPSE was detected by Western blotting. Based on the detection results, transfection was amplified in 10 cm culture dishes at the optimal dosage ratio. After culturing for 72 hours, the culture medium was collected, and cell debris was removed by centrifugation at 500×g for 5 min. The supernatant was collected to obtain the WJS HPSE recombinant baculovirus solution (P1 generation). The multiplicity of infection (MOI) of the P1 generation virus was calculated using the formula "Virus titer (pfu / mL) = (number of plaques x virus dilution factor) / volume of virus infected per well" through viral plaque assay. Based on the P1 generation virus titer results, SF9 cells in 10 cm culture dishes were infected with the P1 generation virus solution with MOI=0.1 to amplify and obtain the P2 generation virus. After determining the P2 generation virus titer through viral plaque assay, SF9 cells were infected with P2 generation virus solutions with different MOI values. After 72 hours of infection, the supernatant was collected, and the expression level of WJS HPSE was detected again by Western blotting. After determining the optimal MOI value for P2 generation virus infection, add an appropriate amount of WJS HPSE P2 virus solution to infect SF9 cells according to this MOI. After 72 hours of infection, collect the cell culture medium, centrifuge at 500×g for 5 min to remove cell debris, and obtain a solution containing WJS HPSE recombinant protein.

[0054] After dialyzing the above protein solution overnight in dialysis buffer (50 mM NaH2PO4, 300 mM NaCl, pH 7.4), the WJS HPSE recombinant protein was purified and enriched using Ni-NTA Beads 6FF packing material: First, the Ni-NTA Beads 6FF column was pre-equilibrated with 5 column volumes of dialysis buffer. Then, the dialyzed WJS HPSE protein solution was added to the nickel column and washed thoroughly with 15–20 column volumes of wash buffer (50 mM NaH2PO4, 300 mM NaCl, pH 7.4) to remove non-specifically bound proteins. Finally, 5 column volumes of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 7.4) were added to elute the target protein. Finally, the eluted WJS HPSE recombinant protein solution was concentrated using Amicon Ultra-15 ultrafiltration centrifuge tubes with a molecular weight cutoff of 10 kDa, and the buffer was replaced with storage buffer (50 mM Tris HCl, 5 mM CaCl2, pH 5.0). The obtained high-purity WJS HPSE recombinant protein was separated by 12% SDS-PAGE gel electrophoresis, and its purification status was detected by Coomassie Brilliant Blue staining. Figure 1The amino acid sequence of heparan sulfate enzyme (WJS HPSE) was finally obtained as shown in SEQ ID NO.2.

[0055] Example 3

[0056] Substrate specificity analysis of recombinant WJS HPSE

[0057] WJS PS polysaccharide (composed of IdoA2S-GlcNAc disaccharide repeating units) isolated and purified from *Solanum lyratum* is the natural substrate of WJS HPSE. After co-incubating WJS HPSE (0.25 μg) with WJS PS (0.20 μg) at 37 ℃ for 24 hours, the enzymatic activity of WJS HPSE was analyzed by 8% TBE-PAGE electrophoresis. Alcian blue and silver nitrate double staining results showed that, compared with the control group (undigested WJS PS), the molecular weight of digested WJS PS in the gel was significantly reduced, indicating that WJS HPSE possesses the activity to cleave WJS PS. Figure 2 (A in the middle).

[0058] Further testing of the enzymatic activity of WJS HPSE against various HS-type glycans was conducted using the same method as described above, and the results are as follows: Figure 2 As shown in Figure A, compared with the control group (which did not undergo WJS HPSE digestion), the molecular weights of K5 polysaccharide (composed of GlcA-GlcNAc disaccharide repetition units), heparin, and HS all decreased significantly after WJS HPSE digestion. However, the migration of HS structural analogs composed of GlcA-GlcNS (K5-NS) and IdoA-GlcNS (Epi-K5) disaccharide repeating units in the gel after digestion showed no significant change compared to the control. These results indicate that WJS HPSE can specifically cleave WJS PS, K5 polysaccharide, heparin, and HS, but has no digestive activity against K5-NS and Epi-K5. Since the HS and heparin glycan structures contain the same disaccharide sequences as K5 polysaccharide, K5-NS, and Epi-K5, it can be inferred that the cleavage target of WJS HPSE for heparin and HS is the glycosidic bond between the GlcA-GlcNAc disaccharides (which have the same disaccharide structure as K5 polysaccharide). Correspondingly, WJS HPSE exhibits significantly less cleavage activity against heparin than HS. This may be because heparin is more sulfated than HS, resulting in a much lower proportion of unmodified GlcA-GlcNAc disaccharide structures in its sugar chains, thus leading to lower cleavage activity of WJS HPSE compared to HS.

[0059] like Figure 2As shown in B, unlike WJS HPSE, human HPSE (hHPSE) has no enzymatic activity against WJS PS and K5 polysaccharides, but it does have enzymatic activity against K5-NS and Epi-K5. Similar to WJS HPSE, hHPSE can also enzymatically cleave heparin and HS, and its cleavage activity against heparin is higher than that against HS.

[0060] Based on the above data, compared with hHPSE, the main substrate cleavage sequence of WJS HPSE provided by this invention is IdoA2S / GlcA-GlcNAc, while the substrate cleavage activity of hHPSE is strictly dependent on... N - Sulfation modification cuts the substrate characteristic sequence into GlcA-GlcNS (such as GlcA-GlcNS6S, etc.).

[0061] It should be noted that the methods for separating and preparing the above polysaccharides K5, K5-NS, and Epi-K5 are referenced in Pinna et al. Antimicrobial Agents and Chemotherapy , (2008) and Hagner-McWhirter et al., Glycobiology ,(2000).

[0062] Example 4

[0063] Enzymatic characterization of recombinant WJS HPSE

[0064] 1. Effects of temperature and pH on enzyme activity

[0065] Using WJS PS (0.2 μg) as substrate, WJS HPSE recombinase (0.25 μg) was added to a reaction buffer of 50 mM Tris-HCl, 5 mM CaCl2·H2O (pH 5.0). The mixture was incubated at 17 ℃, 27 ℃, 37 ℃, 47 ℃, 57 ℃, and 67 ℃ for 3 hours, respectively. TBE-PAGE electrophoresis and Alcian blue-silver nitrate double staining analysis showed that... Figure 3 (A) In all temperature groups, the migration rate of WJS PS bands after enzyme digestion was higher than that of the control group without enzyme. The degradation rate and migration rate increased most significantly under the conditions of 57 ℃-67 ℃, indicating that this temperature range is the peak range of enzyme digestion efficiency. In addition, the 67 ℃ treatment group still maintained significant enzyme digestion activity, confirming that the enzyme has high temperature stability.

[0066] The effect of pH (3.0–9.0) on enzyme activity was investigated at the optimum temperature of 57 °C. WJS HPSE (0.25 μg) and WJS PS (0.2 μg) were added to 50 mM Tris-HCl and 5 mM CaCl2·H2O. A separate WJS PS control was included in each group to eliminate the influence of pH on the substrate itself. After 3 hours of incubation, TBE-PAGE electrophoresis and Alcian blue-silver nitrate double staining analysis showed (…). Figure 3 The enzymatic activity of WJS HPSE (as shown in section B) exhibits a significant pH dependence. The enzymatic activity of WJS HPSE is strongest at pH 4.0. When the pH is above 7.0, there is no difference in migration rate between the enzymatically digested group and the control group, indicating complete loss of enzyme activity, confirming that the catalytic function of WJS HPSE is strictly dependent on an acidic environment.

[0067] 2. Effects of metal ions and anions in the buffer system on WJS HPSE enzyme activity

[0068] Metal ions, acting as cofactors for enzymes, typically have a significant impact on enzyme activity. To investigate the effects of different metal ions on WJS HPSE activity, this study added 5 mM of different metal ions to a reaction buffer (50 mM Tris-HCl) at pH 4.0. WJS HPSE (0.25 μg) was then used to digest WJS PS (0.2 μg) at 57 °C for 3 h. To eliminate the interference of structural changes in WJS PS under different metal ion buffers on the experimental results, a separate control was set up for each experiment. Figure 4 As shown in Figure A, in the presence of Zn 2+ Cu 2+ or Ni 2+ In the reaction buffer, the migration positions of the enzyme-digested WJS PS in the gel were significantly lower than those of the control, indicating that under the above conditions, the efficiency of WJS HPSE digestion of WJS PS was significantly enhanced. Among them, Cu... 2+ The promoting effect on WJS HPSE was the strongest, followed by Zn. 2+ and Ni 2+ In the presence of Li + K + Ca 2+ Na + or Mg 2+ In the reaction buffer, the migration of WJS PS after digestion in the gel was slightly downward compared with the control, indicating that WJS HPSE has weak digestion activity in these five ion buffers.

[0069] To investigate the effect of anions on the activity of WJS HPSE, reaction buffers with different sodium salts (50 mM, pH=4.0) were prepared. WJS PS (0.2 μg) was digested with WJS HPSE (0.25 μg) at 57 °C and incubated for 3 h. To eliminate the interference of structural changes in WJS PS in different anion buffers on the experimental results, a separate control was set up for each experiment. Figure 4 As shown in B, in the presence of F - H2PO4 2- HPO4 2- and CH3COO - In the reaction buffer, the migration positions of the enzyme-digested WJS PS in the gel were significantly lower than those of the control, indicating that these four anions significantly promoted the activity of WJS HPSE. Among them, H2PO4... 2- and HPO4 2- The most significant promoting effect was observed in the digestion of WJS PS by WJS HPSE. In the presence of N... 3- In the reaction buffer, the digested WJS PS shifted slightly downwards in the gel compared to the control, indicating that WJS HPSE had weaker digestion activity under these conditions. However, in the reaction buffer containing Br... - Cl - SO4 2- NO 3- I - SCN - and CO3 2- In the reaction buffer, the migration position of the enzyme-digested WJS PS in the gel did not change significantly compared with the control group, indicating that WJS HPSE had no enzyme digestion activity under the above conditions.

[0070] Example 5

[0071] use QuickCut Xho I and QuickCut BamH I Enzymes (Takara Bio Engineering (Dalian) Co., Ltd.) contain... Xho I The WJS HPSE sequence (synthesized by Qingke Biotechnology Co., Ltd.) containing the BamHI restriction site was double-digested with the pLVX-IRES-Puro vector (reacted at 37 ℃ for 60 minutes). The reaction system is shown in Table 3:

[0072] Table 3. Double enzyme digestion reaction system

[0073]

[0074] After separation and purification of the enzyme digestion products by agarose gel electrophoresis, the WJS HPSE fragment was ligated to the linearized pLVX-IRES-Puro vector using T4 DNA ligase. The ligation product was transformed into DH5α competent cells and plated on LB solid medium containing ampicillin. After overnight incubation at 37 °C, single colonies were picked and inoculated into 5 mL of LB liquid medium containing ampicillin resistance, and cultured at 37 °C with shaking until the OD600 nm value reached 0.6-0.8. 5 µL of this bacterial culture was transferred to 50 mL of LB liquid medium containing ampicillin resistance and cultured at 37 °C with shaking for 12-16 hours. Plasmids were extracted from the amplified bacterial culture using the Endo-Free Plasmid Mini Kit II (Omega Bio-Tek). The recombinant plasmid was digested and verified using the same double enzyme digestion system as described above. Agarose gel electrophoresis showed the expected size fragment (WJS HPSE insert and pLVX-IRES-Puro vector backbone), preliminarily confirming successful construction. Further DNA sequencing (Qingke Biotechnology Co., Ltd.) confirmed the sequence was correct, and the pLVX-IRES-Puro-WJS HPSE lentiviral expression vector was finally obtained.

[0075] Reference to literature method (Fang) et al. , Scientific Reports (2016) To construct a stable HEK293 cell line expressing WJS HPSE. First, HEK293T cells were seeded in 10 cm culture dishes and cultured until 80% confluence was achieved. The pLVX-IRES-Puro-WJS HPSE, psPAX2, and pMD2.G plasmids were co-transfected into HEK293T cells, gently mixed, and cultured at 37 °C for 6 hours. Then, 20 mL of fresh complete medium was added, and the cells were cultured for another 48 hours. The first batch of lentiviral supernatant was collected (temporarily stored at 4 °C). After adding 20 mL of medium and culturing for 24 hours, the second batch of supernatant was collected and mixed with the first batch. HEK293 cells were infected with concentrated lentivirus for 24 hours, then cultured in selection medium containing 1.75 mg / mL puromycin for 48 hours. The surviving cells were expanded to obtain stable HEK293 cells expressing WJS HPSE. WJS HPSE Cell line.

[0076] Reference to literature method (Fang) et al. , Scientific Reports (2016) Extraction of wild-type HEK293 (HEK293) 对照 ) and HEK293 WJS HPSE Endogenous HS in cells, TBE-PAGE electrophoresis and Alcian blue-silver nitrate double staining analysis showed ( Figure 5 ), compared with control cells HEK293 对照In comparison, HEK293, which expresses WJS HPSE, WJS HPSE The molecular weight of endogenous HS glycans was significantly reduced, confirming that WJS HPSE can effectively degrade HS glycans in cells.

[0077] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A heparan sulfate enzyme, characterized in that, The amino acid sequence of the heparan sulfate enzyme is shown in SEQ ID NO.

2.

2. The gene encoding the heparan sulfate enzyme of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the gene described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector is obtained by cloning the gene as described in claim 2 into the expression vector pFastBac1.

5. A recombinant bacterial strain, characterized in that, The recombinant strain includes the gene of claim 2 or the recombinant expression vector of any one of claims 3-4.

6. A method for preparing heparanase sulfate, characterized in that, Includes the following steps: The recombinant expression vector described in claim 3 or 4 was used to transform DH10Bac competent cells, and the cells were purified to obtain the recombinant plasmid. The recombinant plasmid was transfected into SF9 cells and placed in cell culture medium for protein expression. Then, the protein was purified using affinity chromatography to obtain the heparan sulfate enzyme.

7. The use of the heparanase of claim 1 in the preparation of low molecular weight heparan sulfate.

8. The use of the heparan sulfate enzyme of claim 1 in the glycan desorption of K5 polysaccharide, heparin or heparan sulfate.

9. A method for reducing the molecular weight of endogenous heparan sulfate glycans in cells, characterized in that, The method includes the following steps: The gene described in claim 2 was cloned into the pLVX-IRES-Puro vector, and the ligation product was transformed into DH5α competent cells to construct the pLVX-IRES-Puro-WJS HPSE lentiviral expression vector. The pLVX-IRES-Puro-WJS HPSE lentiviral expression vector was co-transfected with psPAX2 plasmid and pMD2.G plasmid into HEK293T cells to obtain lentivirus. HEK293 cells were then infected with lentivirus and cultured to obtain HEK293 cells stably expressing heparan sulfate enzyme. WJS HPSE Cell line.

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