A hyaluronic acid-binding protein polymer and its application in detecting hyaluronic acid

Abstract

The present invention discloses a hyaluronic acid-binding protein dimer and its application in detecting hyaluronic acid. The dimer protein comprises two monomers, wherein each monomer comprises a first protein functional region and a second protein functional region, and the first protein functional region contains the sequence shown in SEQ ID NO: 1; the first protein functional region forms a dimer form through the second protein functional region. The VG1 dimer protein of the present invention has a higher affinity than the VG1 monomer and the existing TSG-6-Fc, and can be applied to the detection of hyaluronic acid.

Description

Technical Field

[0001] The present invention relates to the field of biochemical detection, and particularly relates to a hyaluronic acid-binding protein aggregate and its application in detecting hyaluronic acid. Background Art

[0002] Hyaluronic acid (HA) is a macromolecular glycosaminoglycan with a simple structure but complex functions. It is usually composed of 2000 - 2500 glucuronic acid-(β1-3)-N-acetylglucosamine disaccharide units linked by β1-4 glycosidic bonds to form a polymer with a molecular weight of about 106 - 107. Hyaluronic acid is widely distributed in the biological world. In the human body, hyaluronic acid mainly exists in parts such as the skin, heart, synovial fluid of joints, and bones, and has various functions such as space filling and lubrication. In malignant tumors, hyaluronic acid participates in the proliferation, adhesion, migration of tumor cells and induction of multi-drug resistance of tumors, and plays an important role in tumor development and metastasis.

[0003] HA is an important diagnostic indicator for many diseases. HA can reflect the function and damaged degree of liver endothelial cells. There is a close relationship between the increased level of HA in liver cirrhosis and the degree of pathological changes in liver tissue. When the HA level reaches 250 μg / L, liver cirrhosis can be diagnosed; when the HA level reaches 165 μg / L, it can be used as the boundary between chronic active hepatitis and chronic persistent hepatitis; the serum HA level in acute hepatitis may have a slight increase. In liver cancer, HA is significantly increased. The serum HA level in chronic nephritis and chronic renal insufficiency is significantly increased and is positively correlated with creatinine and urea nitrogen. The serum HA level in pulmonary mesothelioma is significantly increased. As one of the markers for bladder tumors, the content of HA in urine is detected to assist in the diagnosis of bladder tumors. HA can also be applied to the diagnosis of psoriasis. In addition, recent studies have confirmed that hyaluronic acid has an overexpression phenomenon at the tumor site, so hyaluronic acid can also be used as a marker for malignant tumors for tumor diagnosis.

[0004] There are already various HA detection and diagnostic reagents on the market, and the active ingredient for detecting HA is hyaluronic acid-binding protein (HABP). HABP is conventionally Versican protein extracted from bovine nasal cartilage or a Versican protein fragment expressed by Escherichia coli. In 2005, Seyfried first reported the recombinant expression of human Versican G1 domain (1-350aa of NP_001157569.1) using the Drosophila expression system and experimentally confirmed its hyaluronic acid-binding function (Seyfried, McVey et al. 2005). In 2011, Clark expressed the VG1 protein with the same base sequence as Seyfried in the Escherichia coli BL21(DE3)pLysS cells system, but it was in the form of inclusion bodies and had low sensitivity for HA detection.

[0005] CN104093415B discloses another recombinant HA-binding protein, however, this functional sequence is from TSG-6. In existing clinical diagnoses, the commonly used HABP is conventionally Versican protein extracted from bovine nasal cartilage or the Versican protein fragment VG1 expressed by Escherichia coli. The monomeric affinity of the existing VG1 protein is low, and there is a lack of a VG1 protein with high affinity. Summary of the Invention

[0006] In view of the above-mentioned defects in the prior art, the object of the present invention is to provide a new hyaluronic acid detection protein and its uses. The inventors of the present invention unexpectedly found during the development of recombinant hyaluronidase-binding protein that when a fusion protein (VG1-Fc) is formed by expressing VG1 and Fc, it has higher affinity and better specificity than TSG-6-linker module (LM). On the other hand, due to the high affinity and good specificity of VG1-Fc, the sensitivity of commercially available kits and the prior art TSG-6-linker module can be achieved without Biotin labeling. The kit developed based on the VG1-Fc protein can effectively detect HA in, for example, blood and tissues, providing a new option for HA-related research.

[0007] One of the technical solutions of the present invention for the defects of the prior art is: a dimeric protein that binds to hyaluronic acid, the dimeric protein comprising 2 monomers, wherein each monomer comprises a first protein functional region and a second protein functional region, the first protein functional region comprises the sequence shown in SEQ ID NO:1, or a sequence having 85%, 90%, 95%, 98% or more than 99% identity therewith; the first protein functional region forms a dimeric form through the second protein functional region.

[0008] The two monomers may be the same or different, where the two first protein functional regions may be the same or different, and the two second protein functional regions may be the same or different; when they are different, the amino acid sequences of the two protein functional regions have an identity of 85%, 90%, 95%, 98% or more than 99%.

[0009] Preferably, the second protein functional region contains the sequence shown in SEQ ID NO:2. More preferably, the second protein functional region further contains the sequence shown in SEQ ID NO:8.

[0010] Preferably, the monomer contains the sequence shown in SEQ ID NO:3. More preferably, the monomer contains the sequence shown in SEQ ID NO:4.

[0011] To solve the above technical problems, the second technical solution of the present invention is: a nucleic acid molecule encoding a monomer of a dimer protein that binds to hyaluronic acid as described in any one of the above. Preferably, the nucleic acid molecule contains the sequences shown in SEQ ID NO:5 and SEQ ID NO:6.

[0012] To solve the above technical problems, the third technical solution of the present invention is: an expression vector that contains the nucleic acid molecule as described above.

[0013] To solve the above technical problems, the fourth technical solution of the present invention is: a prokaryotic or eukaryotic host cell that contains the expression vector as described above. Preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

[0014] To solve the above technical problems, the fifth technical solution of the present invention is: the application of the dimer protein, the nucleic acid molecule, the expression vector or the host cell as described in any one of the above in the preparation of in vivo diagnostic and therapeutic pharmaceutical preparations and / or pharmaceutical compositions for diseases.

[0015] To solve the above technical problems, the sixth technical solution of the present invention is: a detection kit that includes the hyaluronic acid-binding protein as described above.

[0016] Preferably, the detection kit contains a hyaluronic acid degrading enzyme. More preferably, the hyaluronic acid degrading enzyme is hyaluronidase. Even more preferably, the hyaluronidase is Hyal1, Hyal2, Hyal3, mammalian testicular hyaluronidase, hyaluronidase fusion protein, variants of the hyaluronidase and / or modified products of the variants of the hyaluronidase. Further preferably, the hyaluronidase is human testicular hyaluronidase, and the human testicular hyaluronidase is PH20 with a sequence as shown in SEQ ID NO:9.

[0017] The kit is applied to the detection of hyaluronic acid in tissue or fluid samples, and the tissue is derived from species such as humans and mice. The tissue is preferably human stromal tissue. The human stromal tissue is more preferably a stromal tissue sample from a tumor. The fluid includes blood, serum, urine, sweat, semen, saliva, cerebrospinal fluid or lymph.

[0018] To solve the above technical problems, the seventh technical solution of the present invention is: a method for detecting hyaluronic acid, which includes the following steps:

[0019] (1) Take a sample containing hyaluronic acid; the sample is a stromal tissue sample or a fluid sample, the stromal tissue is preferably a stromal tissue sample from a tumor, and the fluid sample includes blood, serum, urine, sweat, semen, saliva, cerebrospinal fluid and / or lymph sample;

[0020] (2) Detect the sample using the detection kit as described above. The method for detecting hyaluronic acid preferably further includes a step of developing color with a chromogenic solution. The chromogenic solution is preferably DAB.

[0021] Preferably, the method for detecting hyaluronic acid further includes step (3) quantitatively or semi-quantitatively analyzing the content of hyaluronic acid in the sample according to the detection result.

[0022] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be arbitrarily combined to obtain various preferred examples of the present invention.

[0023] The reagents and raw materials used in the present invention are all commercially available.

[0024] The positive and progressive effects of the present invention are as follows:

[0025] The present invention provides a novel hyaluronic acid-binding protein and provides its application in detecting and quantifying hyaluronic acid in biological samples. The kit containing this protein can be used to diagnose and predict the disease development, treatment effect and prognosis of patients.

[0026] The obtained hyaluronic acid-binding protein fragment of the present invention can conveniently remove the Fc fragment. As an in vitro diagnostic reagent, it does not precipitate antigens and can reduce the false positive rate. The obtained hyaluronic acid-binding protein fragment has a high affinity for hyaluronic acid. The dimer of the hyaluronic acid-binding protein has a much higher affinity for hyaluronic acid than the prior art and has higher detection sensitivity. As an in vivo diagnostic reagent, after being labeled with a radionuclide, the hyaluronic acid-binding protein fragment has a shorter half-life in normal tissues than the full-length antibody and can be rapidly degraded, which is beneficial to reducing the damage to normal tissues of the body caused by the Fc effector function. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the KJ-V01 expression vector.

[0028] Figure 2 When coated with HA, each binding protein was serially diluted and loaded, and the OD450 curve graph was plotted based on the detection data of the corresponding enzyme-linked antibody.

[0029] Figure 3 When coated with HA, each binding protein was serially diluted and loaded, and the OD450 curve graph was plotted based on the detection data of the corresponding enzyme-linked antibody.

[0030] Figure 4 When coated with HA, each binding protein was serially diluted and loaded, and the OD450 curve graph was plotted based on the detection data of the corresponding enzyme-linked antibody.

[0031] Figure 5 Experimental result graph of the application of specific proteins to skin tissue immunohistochemistry for VG1-Fc. Detailed implementation manners

[0032] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0033] Example 1. Expression of VG1-Fc protein

[0034] Entrust a gene synthesis company to optimize the codons for the provided amino acid sequences and synthesize the DNA fragments encoding VG1-Fc (SEQ ID NO:5, SEQ ID NO:6), VG1-His, and TSG-6-Fc in full gene, and clone them into the KJ-V01 expression vector constructed by the inventor ( Figure 1 ), and the expression vector consists of the following elements:

[0035] 1) Glutamine synthetase gene, as a selection marker,

[0036] 2) Replication origin, ori,

[0037] 3) Replication origin from vector pUC18, which allows this plasmid to replicate in Escherichia coli,

[0038] 4) β-lactamase gene, which confers ampicillin resistance in Escherichia coli,

[0039] 5) Immediate early enhancer and promoter from human cytomegalovirus,

[0040] 6) Human 1-immunoglobulin polyadenylation (“poly A”) signal sequence.

[0041] As described above, the full-length genes of VG1-Fc (SEQ ID NO:4), VG1-His (SEQ ID NO:7), and TSG-6-Fc (see CN104093415B, SEQ ID NO:215) were synthesized, digested with EcoRI and HindIII, and then cloned into the KJ-V01 vector digested with the same enzymes, finally constructing VG1-Fc / KJ-V01, VG1-His / KJ-V01, and TSG-6-Fc / KJ-V01.

[0042] The constructed expression vectors of VG1-Fc / KJ-V01, VG1-His / KJ-V01, and TSG-6-Fc / KJ-V01 were transformed into Escherichia coli TOP10, and positive clones were picked and inoculated into 100 ml of LB medium for amplification. Transfection-grade DNA was prepared using the Plasmid Plus Midi Kit (cat: 12943) from Qiagen. The obtained plasmid was linearized by single digestion with ScaI, and then the digested plasmid was purified by phenol-chloroform extraction and ethanol precipitation (the method is from "Molecular Cloning: A Laboratory Manual, Third Edition"). The plasmid DNA was transfected into CHO-K1 (Chinese hamster ovary cells, purchased from ATCC) using the liposome method kit from Gibco, and the operation was carried out according to the manufacturer's instructions.

[0043] At 24 - 48 hours after transfection, the cells were inoculated into a 96-well plate at a certain cell density, and the cell medium was a selection medium containing a selection drug. After static culture for 3 - 4 weeks, clones gradually grew out. Larger monoclonal clones were selected, the medium was changed, and the cells were dispersed. After 24 hours, the supernatant was taken for detection of VG1-Fc / KJ-V01, VG1-His / KJ-V01, and TSG-6-Fc / KJ-V01. HA was coated on an ImmunoX ELISA plate, and the clones with higher response values were successively amplified to a 24-well plate, a T25 culture flask, a T75 culture flask, and a 125 mL shake flask. Clones with good growth were selected for amplification and harvesting of the supernatant. The supernatant after harvesting was purified using a ProA column or a nickel column. The purified protein was stored in a PBS system with a pH of 7.4.

[0044] SEQ ID NO:1

[0045] LHKVKVGKSPPVRGSLSGKVSLPCHFSTMPTLPPSYNTSEFLRIKWSKIEVDKNGKDLKETTVLVAQNGNIKIGQDYKGRVSVPTHPEAVGDASLTVVKLLASDAGLYRCDVMYGIEDTQDTVSLTVDGVVFHYRAATSRYTLNFEAAQKACLDVGAVIATPEQLFAAYEDGFEQCDAGWLADQTVRYPIRAPRVGCYGDKMGKAGVRTYGFRSPQETYDVYCYVDHLDGDVFHLTVPSKFTFEEAAKECENQDARLATVGELQAAWRNGFDQCDYGWLSDASVRHPVTVARAQCGGGLLGVRTLYRFENQTGFPPPDSRFDAYCFK

[0046] SEQ ID NO:2

[0047] CPPCPAPELLG

[0048] SEQ ID NO:3

[0049] LHKVKVGKSPPVRGSLSGKVSLPCHFSTMPTLPPSYNTSEFLRIKWSKIEVDKNGKDLKETTVLVAQNGNIKIGQDYKGRVSVPTHPEAVGDASLTVVKLLASDAGLYRCDVMYGIEDTQDTVSLTVDGVVFHYRAATSRYTLNFEAAQKACLDVGAVIATPEQLFAAYEDGFEQCDAGWLADQTVRYPIRAPRVGCYGDKMGKAGVRTYGFRSPQETYDVYCYVDHLDGDVFHLTVPSKFTFEEAAKECENQDARLATVGELQAAWRNGFDQCDYGWLSDASVRHPVTVARAQCGGGLLGVRTLYRFENQTGFPPPDSRFDAYCFKRRMCDKTHTCPPCPAPELLG

[0050] SEQ ID NO:4

[0051] LHKVKVGKSPPVRGSLSGKVSLPCHFSTMPTLPPSYNTSEFLRIKWSKIEVDKNGKDLKETTVLVAQNGNIKIGQDYKGRVSVPTHPEAVGDASLTVVKLLASDAGLYRCDVMYGIEDTQDTVSLTVDGVVFHYRAATSRYTLNFEAAQKACLDVGAVIATPEQLFAAYEDGFEQCDAGWLADQTVRYPIRAPRVGCYGDKMGKAGVRTYGFRSPQETYDVYCYVDHLDGDVFHLTVPSKFTFEEAAKECENQDARLATVGELQAAWRNGFDQCDYGWLSDASVRHPVTVARAQCGGGLLGVRTLYRFENQTGFPPPDSRFDAYCFKRRMCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0052] SEQ ID NO:5

[0053] CTGCACAAGGTGAAAGTGGGCAAATCCCCTCCAGTGAGGGGATCTCTGAGCGGCAAAGTGAGCCTCCCTTGTCACTTCAGCACAATGCCTACTCTGCCTCCAAGCTACAACACTAGCGAGTTTCTGAGGATCAAGTGGTCCAAGATCGAGGTCGACAAGAACGGAAAGGATCTGAAGGAGACAACTGTGCTCGTCGCTCAGAACGGCAACATCAAGATCGGCCAAGATTATAAAGGCAGAGTCTCCGTGCCAACACATCCAGAGGCTGTGGGCGATGCCTCTCTGACAGTGGTCAAGCTGCTGGCTAGCGACGCCGGACTGTATAGGTGTGACGTGATGTACGGCATCGAAGACACACAAGATACTGTGAGCCTCACAGTCGATGGCGTGGTGTTCCACTATAGGGCTGCCACTTCTAGGTACACACTCAACTTCGAGGCCGCTCAGAAAGCTTGTCTCGACGTGGGCGCTGTGATCGCTACTCCAGAGCAGCTGTTCGCCGCCTACGAGGACGGCTTCGAGCAATGTGATGCCGGCTGGCTCGCCGACCAGACTGTGAGATACCCTATTAGGGCCCCTAGAGTCGGATGCTACGGCGACAAAATGGGAAAGGCCGGCGTGAGAACTTACGGATTTAGGAGCCCACAAGAGACTTATGACGTCTACTGCTACGTGGACCATCTGGATGGCGATGTCTTCCATCTGACAGTGCCATCCAAGTTCACTTTCGAGGAGGCCGCCAAGGAATGCGAAAACCAAGATGCTAGGCTGGCCACAGTCGGAGAACTGCAAGCCGCTTGGAGGAACGGCTTCGATCAGTGCGACTATGGCTGGCTCTCCGATGCTAGCGTCAGACACCCAGTGACAGTGGCTAGAGCTCAGTGTGGAGGAGGACTCCTCGGCGTGAGGACTCTGTATAGGTTCGAGAACCAGACTGGCTTCCCTCCTCCAGATTCTAGGTTCGACGCCTACTGTTTCAAG

[0054] SEQ ID NO:6

[0055] TGCCCACCTTGTCCCGCCCCTGAACTGCTGGGC

[0056] SEQ ID NO:7

[0057] GKVSLPCHFSTMPTLPPSYNTSEFLRIKWSKIEVDKNGKDLKETTVLVAQNGNIKIGQDYKGRVSVPTHPEAVGDASLTVVKLLASDAGLYRCDVMYGIEDTQDTVSLTVDGVVFHYRAATSRYTLNFEAAQKACLDVGAVIATPEQLFAAYEDGFEQCDAGWLADQTVRYPIRAPRVGCYGDKMGKAGVRTYGFRSPQETYDVYCYVDHLDGDVFHLTVPSKFTFEEAAKECENQDARLATVGELQAAWRNGFDQCDYGWLSDASVRHPVTVARAQCGGGLLGVRTLYRFENQTGFPPPDSRFDAYCFKPKEATHHHHHH

[0058] SEQ ID NO:8

[0059] RRMCDKTHT

[0060] SEQ ID NO:9

[0061] LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY

[0062] Example 2. Affinity Test of VG1-Fc Dimer Protein and Monomeric VG1 (His-tag) Protein

[0063] Take 1 mg / ml hyaluronic acid solution, dilute it 10-fold with 0.5 mol / L sodium carbonate solution (pH 9.6), mix well, add 100 μl per well into a 96-well plate, and coat overnight at 2 - 8 °C. Dilute VG1-Fc dimer protein and VG1 (His-tag) protein starting from 1000 ng / ml, then dilute 10-fold to 100 ng / ml, and then continue to perform 5 two-fold serial dilutions to obtain a total of 7 concentration points, which are added to the 96-well plate coated with hyaluronic acid, 100 μl per well, and incubated at 37 °C for about 1 hour. VG1-Fc is detected with anti-Human IgG Fcγ-HRP (ThermoFisher Scientific) enzyme-linked antibody, and VG1 (His-tag) is detected with Anti-6X His Antibody (HRP) (Abcam) enzyme-linked antibody. Then, develop color with TMB substrate, and measure the absorbance value with a Molecular Devices, Spectra Max M2e microplate reader at 450 / 630 nm. Compared with the monomeric VG1 (His-tag) protein, the VG1-Fc dimer protein significantly exhibits excellent affinity on the HA-coated plate ( Figure 2 ).

[0064] Example 3. Affinity Test of VG1-Fc Dimer Protein and TSG-6-Fc Dimer Protein

[0065] Take 1 mg / ml hyaluronic acid solution, dilute it 10 times with 0.5 mol / L sodium carbonate solution (pH 9.6), mix well, add 100 μl per well into a 96-well plate, and coat overnight at 2 - 8 °C. Dilute VG1-Fc dimer protein and TSG-6-Fc dimer protein starting from 10 nM, then dilute 10-fold to 1 nM, and then continue to dilute in a 2-fold serial dilution for 5 points, with a total of 7 concentration points added to the 96-well plate coated with hyaluronic acid, 100 μl per well, and incubate at 37 °C for about 1 hour. Detect with anti-Human IgG Fcγ-HRP (Thermo Fisher Scientific) enzyme-linked antibody, then develop color with TMB substrate, and measure the absorbance value with a Molecular Devices, Spectra Max M2e microplate reader at 450 / 630 nm. Compared with monomeric TSG-6-Fc, VG1-Fc dimer protein significantly shows higher affinity on the HA-coated plate. The EC50 value of VG1-Fc is 0.38 ± 0.04 nM, and the EC50 value of TSG-6-Fc is 0.91 ± 0.11 nM, P < 0.01( Figure 3 ).

[0066] Example 4. VG1-Fc Has Higher Affinity Compared with TSG-6-Fc Dimer Protein

[0067] In this example, the affinity of VG1-Fc and TSG-6-Fc was further evaluated under the same detection conditions as in patent application CN104093415B. Direct conjugation of VG1-Fc and TSG-6-Fc proteins with the primary amine activator NHS-PEG4-biotin (Thermo Fisher Scientific) was carried out according to the kit instructions. Take 200 μl of 1 mg / ml protein in PBS system and incubate with the corresponding volume of 20 mM NHS-PEG4-biotin reagent on ice for 2 hours or at room temperature for 30 - 60 minutes, and then purify and collect the labeled protein through the Zeba TM Spin Desalting Column in the kit. The N-hydroxysuccinimide ester (NHS) group of NHS-PEG4-biotin specifically and efficiently reacts with lysine and N-terminal amino acids at pH 7 - 9 to form stable amide bonds. The hydrophilic polyethylene glycol (PEG) spacer arm imparts water solubility to the biotinylated molecule, reducing the aggregation of biotinylated proteins stored in solution. The PEG spacer arm can also provide a long and flexible connection for the biotinylation reagent, minimizing the steric hindrance associated with the binding of the biotinylated protein molecule.

[0068] Take 1 mg / ml hyaluronic acid solution and dilute it 10-fold with 0.5 mol / L sodium carbonate solution (pH 9.6). Mix well and add 100 μl per well into a 96-well plate. Coat overnight at 2 - 8°C. Biotinylated VG1 dimer protein and TSG-6-Fc protein are diluted starting from 10 nM, then serially diluted 10-fold to 1 nM, and further serially diluted 2-fold for 5 points, resulting in a total of 7 concentration points, which are added to the 96-well plate coated with hyaluronic acid, 100 μl per well, and incubated at 37°C for about 1 hour. Detect with HRP-labeled Streptavidin (Beyotime Biotechnology) enzyme-linked antibody, then develop color with TMB substrate, and measure the absorbance value at 450 / 630 nm using a Molecular Devices, Spectra Max M2e microplate reader. Biotinylated VG1-Fc still showed significantly higher affinity than biotinylated TSG-6-Fc on the HA-coated plate. At a concentration of 1 nM, the OD450nm values of VG1-Fc and TSG-6-Fc proteins were 0.99 ± 0.01 and 0.14, respectively, P < 0.01( Figure 4 ).

[0069] Example 5. Immunohistochemical application of VG1 dimer protein

[0070] Detect the expression of HA in normal human skin tissue by immunohistochemistry. Dewax and rehydrate paraffin-embedded sections of normal human skin tissue. First, place the sections in xylene for 10 minutes, twice, and then successively in 100%, 95%, 85%, and 75% ethanol, for 5 - 10 minutes at each step. Then wash with distilled water for 5 minutes. Place the sections in citrate buffer (pH 6.0) and heat for antigen retrieval for 8 minutes, and cool naturally to room temperature. Rinse three times with PBS, 3 minutes each time. Use 3% H 2 O 2 Incubate for 15 minutes to block endogenous peroxidase, and then rinse three times with PBS, 3 minutes each time.

[0071] Three experimental groups were set up: the PH20 pretreatment group, the normal experimental group, and the negative control group. The sections of the PH20 pretreatment group were placed in a solution of recombinant human PH20 (Rhinobio) diluted to approximately 90 units / ml with enzyme diluent and incubated at 37°C for 2 hours; the other normal experimental group and the negative control group were directly placed in the enzyme diluent and incubated at 37°C for 2 hours. After incubation, 50 - 100 μl of goat serum was added dropwise and incubated in an incubator at 37°C for 20 minutes, and then centrifuged dry without washing. The sections of the PH20 pretreatment group were added dropwise with a 20 μg / ml VG1 dimer protein solution, the normal experimental group was added dropwise with different concentrations of VG1 solutions of 20, 2, 0.2, and 0.04 μg / ml, and the negative control group was added dropwise with an equal amount of PBS, incubated overnight in a refrigerator at 4°C, and rewarmed in an incubator at 37°C for 1.5 hours. All sections were taken out of the incubator, rinsed three times with PBS for 3 minutes each time, added dropwise with 50 - 100 μl of HRP-labeled Streptavidin (Beyotime Biotechnology), and incubated in an incubator at 37°C for 20 minutes. Rinsed three times with PBS for 3 minutes each time, and then developed color with DAB chromogenic solution (AURAGENE), incubated at room temperature for 1 - 5 minutes, and controlled the reaction time under the microscope. The developed slides were washed with distilled water, immersed in hematoxylin for counterstaining for 3 - 5 seconds, rinsed with distilled water, and blued with PBS. Dehydrated with alcohols of various grades (60 - 100%) for 5 minutes each. After taking out, placed in xylene for 10 minutes, twice, sealed with neutral gum, observed under a microscope, photographed, and the results are as Figure 5 shown.

[0072] By the above immunohistochemical method, the appropriate concentration range of the VG1 dimer protein was selected, and its optimal working concentration was shown to be 0.2 - 2 μg / ml.

[0073] In summary, it can be seen that the VG1-Fc dimer protein and the VG1 dimer protein obtained in the present invention have a higher affinity for hyaluronic acid than the prior art, and at the same time, they also show better detection effects in immunohistochemical experiments.

[0074] The applicant declares that the present invention uses the above embodiments to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention. SEQUENCE LISTING <110> Suzhou Ruian Biotechnology Co., Ltd. <120> A hyaluronic acid-binding protein aggregate and its application in detecting hyaluronic acid <130> P20010869C <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 327 <212> PRT <213> Homo sapiens <400> 1 Leu His Lys Val Lys Val Gly Lys Ser Pro Pro Val Arg Gly Ser Leu 1 5 10 15 Ser Gly Lys Val Ser Leu Pro Cys His Phe Ser Thr Met Pro Thr Leu 20 25 30 Pro Pro Ser Tyr Asn Thr Ser Glu Phe Leu Arg Ile Lys Trp Ser Lys 35 40 45 Ile Glu Val Asp Lys Asn Gly Lys Asp Leu Lys Glu Thr Thr Val Leu 50 55 60 Val Ala Gln Asn Gly Asn Ile Lys Ile Gly Gln Asp Tyr Lys Gly Arg 65 70 75 80 Val Ser Val Pro Thr His Pro Glu Ala Val Gly Asp Ala Ser Leu Thr 85 90 95 Val Val Lys Leu Leu Ala Ser Asp Ala Gly Leu Tyr Arg Cys Asp Val 100 105 110 Met Tyr Gly Ile Glu Asp Thr Gln Asp Thr Val Ser Leu Thr Val Asp 115 120 125 Gly Val Val Phe His Tyr Arg Ala Ala Thr Ser Arg Tyr Thr Leu Asn 130 135 140 Phe Glu Ala Ala Gln Lys Ala Cys Leu Asp Val Gly Ala Val Ile Ala 145 150 155 160 Thr Pro Glu Gln Leu Phe Ala Ala Tyr Glu Asp Gly Phe Glu Gln Cys 165 170 175 Asp Ala Gly Trp Leu Ala Asp Gln Thr Val Arg Tyr Pro Ile Arg Ala 180 185 190 Pro Arg Val Gly Cys Tyr Gly Asp Lys Met Gly Lys Ala Gly Val Arg 195 200 205 Thr Tyr Gly Phe Arg Ser Pro Gln Glu Thr Tyr Asp Val Tyr Cys Tyr 210 215 220 Val Asp His Leu Asp Gly Asp Val Phe His Leu Thr Val Pro Ser Lys 225 230 235 240 Phe Thr Phe Glu Glu Ala Ala Lys Glu Cys Glu Asn Gln Asp Ala Arg 245 250 255 Leu Ala Thr Val Gly Glu Leu Gln Ala Ala Trp Arg Asn Gly Phe Asp 260 265 270 Gln Cys Asp Tyr Gly Trp Leu Ser Asp Ala Ser Val Arg His Pro Val 275 280 285 Thr Val Ala Arg Ala Gln Cys Gly Gly Gly Leu Leu Gly Val Arg Thr 290 295 300 Leu Tyr Arg Phe Glu Asn Gln Thr Gly Phe Pro Pro Pro Asp Ser Arg 305 310 315 320 Phe Asp Ala Tyr Cys Phe Lys 325 <210> 2 <211> 11 <212> PRT <213> Homo sapiens <400> 2 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 <210> 3 <211> 347 <212> PRT <213> Artificial Sequence <220> <223> VG1-Fc <400> 3 Leu His Lys Val Lys Val Gly Lys Ser Pro Pro Val Arg Gly Ser Leu 1 5 10 15 Ser Gly Lys Val Ser Leu Pro Cys His Phe Ser Thr Met Pro Thr Leu 20 25 30 Pro Pro Ser Tyr Asn Thr Ser Glu Phe Leu Arg Ile Lys Trp Ser Lys 35 40 45 Ile Glu Val Asp Lys Asn Gly Lys Asp Leu Lys Glu Thr Thr Val Leu 50 55 60 Val Ala Gln Asn Gly Asn Ile Lys Ile Gly Gln Asp Tyr Lys Gly Arg 65 70 75 80 Val Ser Val Pro Thr His Pro Glu Ala Val Gly Asp Ala Ser Leu Thr 85 90 95 Val Val Lys Leu Leu Ala Ser Asp Ala Gly Leu Tyr Arg Cys Asp Val 100 105 110 Met Tyr Gly Ile Glu Asp Thr Gln Asp Thr Val Ser Leu Thr Val Asp 115 120 125 Gly Val Val Phe His Tyr Arg Ala Ala Thr Ser Arg Tyr Thr Leu Asn 130 135 140 Phe Glu Ala Ala Gln Lys Ala Cys Leu Asp Val Gly Ala Val Ile Ala 145 150 155 160 Thr Pro Glu Gln Leu Phe Ala Ala Tyr Glu Asp Gly Phe Glu Gln Cys 165 170 175 Asp Ala Gly Trp Leu Ala Asp Gln Thr Val Arg Tyr Pro Ile Arg Ala 180 185 190 Pro Arg Val Gly Cys Tyr Gly Asp Lys Met Gly Lys Ala Gly Val Arg 195 200 205 Thr Tyr Gly Phe Arg Ser Pro Gln Glu Thr Tyr Asp Val Tyr Cys Tyr 210 215 220 Val Asp His Leu Asp Gly Asp Val Phe His Leu Thr Val Pro Ser Lys 225 230 235 240 Phe Thr Phe Glu Glu Ala Ala Lys Glu Cys Glu Asn Gln Asp Ala Arg 245 250 255 Leu Ala Thr Val Gly Glu Leu Gln Ala Ala Trp Arg Asn Gly Phe Asp 260 265 270 Gln Cys Asp Tyr Gly Trp Leu Ser Asp Ala Ser Val Arg His Pro Val 275 280 285 Thr Val Ala Arg Ala Gln Cys Gly Gly Gly Leu Leu Gly Val Arg Thr 290 295 300 Leu Tyr Arg Phe Glu Asn Gln Thr Gly Phe Pro Pro Pro Asp Ser Arg 305 310 315 320 Phe Asp Ala Tyr Cys Phe Lys Arg Arg Met Cys Asp Lys Thr His Thr 325 330 335 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 340 345 <210> 4 <211> 558 <212> PRT <213> Artificial Sequence <220> <223> Preferred VG1-Fc <400> 4 Leu His Lys Val Lys Val Gly Lys Ser Pro Pro Val Arg Gly Ser Leu 1 5 10 15 Ser Gly Lys Val Ser Leu Pro Cys His Phe Ser Thr Met Pro Thr Leu 20 25 30 Pro Pro Ser Tyr Asn Thr Ser Glu Phe Leu Arg Ile Lys Trp Ser Lys 35 40 45 Ile Glu Val Asp Lys Asn Gly Lys Asp Leu Lys Glu Thr Thr Val Leu 50 55 60 Val Ala Gln Asn Gly Asn Ile Lys Ile Gly Gln Asp Tyr Lys Gly Arg 65 70 75 80 Val Ser Val Pro Thr His Pro Glu Ala Val Gly Asp Ala Ser Leu Thr 85 90 95 Val Val Lys Leu Leu Ala Ser Asp Ala Gly Leu Tyr Arg Cys Asp Val 100 105 110 Met Tyr Gly Ile Glu Asp Thr Gln Asp Thr Val Ser Leu Thr Val Asp 115 120 125 Gly Val Val Phe His Tyr Arg Ala Ala Thr Ser Arg Tyr Thr Leu Asn 130 135 140 Phe Glu Ala Ala Gln Lys Ala Cys Leu Asp Val Gly Ala Val Ile Ala 145 150 155 160 Thr Pro Glu Gln Leu Phe Ala Ala Tyr Glu Asp Gly Phe Glu Gln Cys 165 170 175 Asp Ala Gly Trp Leu Ala Asp Gln Thr Val Arg Tyr Pro Ile Arg Ala 180 185 190 Pro Arg Val Gly Cys Tyr Gly Asp Lys Met Gly Lys Ala Gly Val Arg 195 200 205 Thr Tyr Gly Phe Arg Ser Pro Gln Glu Thr Tyr Asp Val Tyr Cys Tyr 210 215 220 Val Asp His Leu Asp Gly Asp Val Phe His Leu Thr Val Pro Ser Lys 225 230 235 240 Phe Thr Phe Glu Glu Ala Ala Lys Glu Cys Glu Asn Gln Asp Ala Arg 245 250 255 Leu Ala Thr Val Gly Glu Leu Gln Ala Ala Trp Arg Asn Gly Phe Asp 260 265 270 Gln Cys Asp Tyr Gly Trp Leu Ser Asp Ala Ser Val Arg His Pro Val 275 280 285 Thr Val Ala Arg Ala Gln Cys Gly Gly Gly Leu Leu Gly Val Arg Thr 290 295 300 Leu Tyr Arg Phe Glu Asn Gln Thr Gly Phe Pro Pro Pro Asp Ser Arg 305 310 315 320 Phe Asp Ala Tyr Cys Phe Lys Arg Arg Met Cys Asp Lys Thr His Thr 325 330 335 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 340 345 350 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 355 360 365 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 370 375 380 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 385 390 395 400 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 405 410 415 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 420 425 430 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 435 440 445 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 450 455 460 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 465 470 475 480 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 485 490 495 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 500 505 510 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 515 520 525 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 530 535 540 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 545 550 555 <210> 5 <211> 981 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the first functional region of VG1-Fc <400> 5 ctgcacaagg tgaaagtggg caaatcccct ccagtgaggg gatctctgag cggcaaagtg 60 agcctccctt gtcacttcag cacaatgcct actctgcctc caagctacaa cactagcgag 120 tttctgagga tcaagtggtc caagatcgag gtcgacaaga acggaaagga tctgaaggag 180 acaactgtgc tcgtcgctca gaacggcaac atcaagatcg gccaagatta taaaggcaga 240 gtctccgtgc caacacatcc agaggctgtg ggcgatgcct ctctgacagt ggtcaagctg 300 ctggctagcg acgccggact gtataggtgt gacgtgatgt acggcatcga agacacacaa 360 gatactgtga gcctcacagt cgatggcgtg gtgttccact atagggctgc cacttctagg 420 tacacactca acttcgaggc cgctcagaaa gcttgtctcg acgtgggcgc tgtgatcgct 480 actccagagc agctgttcgc cgcctacgag gacggcttcg agcaatgtga tgccggctgg 540 ctcgccgacc agactgtgag ataccctatt agggccccta gagtcggatg ctacggcgac 600 aaaatgggaa aggccggcgt gagaacttac ggatttagga gcccacaaga gacttatgac 660 gtctactgct acgtggacca tctggatggc gatgtcttcc atctgacagt gccatccaag 720 ttcactttcg aggaggccgc caaggaatgc gaaaaccaag atgctaggct ggccacagtc 780 ggagaactgc aagccgcttg gaggaacggc ttcgatcagt gcgactatgg ctggctctcc 840 gatgctagcg tcagacaccc agtgacagtg gctagagctc agtgtggagg aggactcctc 900 ggcgtgagga ctctgtatag gttcgagaac cagactggct tccctcctcc agattctagg 960 ttcgacgcct actgtttcaa g 981 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the second functional region of VG1-Fc <400> 6 tgcccacctt gtcccgcccc tgaactgctg ggc 33 <210> 7 <211> 321 <212> PRT <213> Artificial Sequence <220> <223> VG1-His <400> 7 Gly Lys Val Ser Leu Pro Cys His Phe Ser Thr Met Pro Thr Leu Pro 1 5 10 15 Pro Ser Tyr Asn Thr Ser Glu Phe Leu Arg Ile Lys Trp Ser Lys Ile 20 25 30 Glu Val Asp Lys Asn Gly Lys Asp Leu Lys Glu Thr Thr Val Leu Val 35 40 45 Ala Gln Asn Gly Asn Ile Lys Ile Gly Gln Asp Tyr Lys Gly Arg Val 50 55 60 Ser Val Pro Thr His Pro Glu Ala Val Gly Asp Ala Ser Leu Thr Val 65 70 75 80 Val Lys Leu Leu Ala Ser Asp Ala Gly Leu Tyr Arg Cys Asp Val Met 85 90 95 Tyr Gly Ile Glu Asp Thr Gln Asp Thr Val Ser Leu Thr Val Asp Gly 100 105 110 Val Val Phe His Tyr Arg Ala Ala Thr Ser Arg Tyr Thr Leu Asn Phe 115 120 125 Glu Ala Ala Gln Lys Ala Cys Leu Asp Val Gly Ala Val Ile Ala Thr 130 135 140 Pro Glu Gln Leu Phe Ala Ala Tyr Glu Asp Gly Phe Glu Gln Cys Asp 145 150 155 160 Ala Gly Trp Leu Ala Asp Gln Thr Val Arg Tyr Pro Ile Arg Ala Pro 165 170 175 Arg Val Gly Cys Tyr Gly Asp Lys Met Gly Lys Ala Gly Val Arg Thr 180 185 190 Tyr Gly Phe Arg Ser Pro Gln Glu Thr Tyr Asp Val Tyr Cys Tyr Val 195 200 205 Asp His Leu Asp Gly Asp Val Phe His Leu Thr Val Pro Ser Lys Phe 210 215 220 Thr Phe Glu Glu Ala Ala Lys Glu Cys Glu Asn Gln Asp Ala Arg Leu 225 230 235 240 Ala Thr Val Gly Glu Leu Gln Ala Ala Trp Arg Asn Gly Phe Asp Gln 245 250 255 Cys Asp Tyr Gly Trp Leu Ser Asp Ala Ser Val Arg His Pro Val Thr 260 265 270 Val Ala Arg Ala Gln Cys Gly Gly Gly Leu Leu Gly Val Arg Thr Leu 275 280 285 Tyr Arg Phe Glu Asn Gln Thr Gly Phe Pro Pro Pro Asp Ser Arg Phe 290 295 300 Asp Ala Tyr Cys Phe Lys Pro Lys Glu Ala Thr His His His His His 305 310 315 320 His <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Partial sequence of the second protein functional region <400> 8 Arg Arg Met Cys Asp Lys Thr His Thr 1 5 <210> 9 <211> 447 <212> PRT <213> Homo sapiens <400> 9 Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp 1 5 10 15 Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro 20 25 30 Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala 35 40 45 Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr 50 55 60 Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro 65 70 75 80 Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile 85 90 95 Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp 100 105 110 Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val 115 120 125 Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu 130 135 140 Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala 145 150 155 160 Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg 165 170 175 Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His 180 185 190 His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile 195 200 205 Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu 210 215 220 Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr 225 230 235 240 Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile 245 250 255 Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val 260 265 270 Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr 275 280 285 Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp 290 295 300 Gly Thr Leu Ser Ile Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp 305 310 315 320 Asn Tyr Met Glu Thr Ile Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu 325 330 335 Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys 340 345 350 Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp 355 360 365 Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly 370 375 380 Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys 385 390 395 400 Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp 405 410 415 Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala 420 425 430 Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr 435 440 445

Claims

1. A dimeric protein conjugated with hyaluronic acid, characterized in that the dimeric protein comprises two monomers, and the amino acid sequence of each monomer is as shown in SEQ ID NO:

4.

2. A nucleic acid molecule encoding a monomer of the dimeric protein conjugated with hyaluronic acid according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that the nucleotide sequence of the nucleic acid molecule contains the sequences shown in SEQ ID NO:5 and SEQ ID NO:

6.

4. An expression vector containing the nucleic acid molecule according to claim 2 or 3.

5. A host cell containing the expression vector according to claim 4.

6. The host cell according to claim 5, characterized in that the host cell is a prokaryotic cell or a eukaryotic cell.

7. Use of the dimeric protein according to claim 1, the nucleic acid molecule according to claim 2 or 3, the expression vector according to claim 4, or the host cell according to claim 5 or 6 in the preparation of a preparation for diagnosing a disease; the disease is a disease related to abnormal HA expression in human skin tissue, liver cancer, lung mesothelial cell carcinoma, bladder tumor, liver cirrhosis, chronic active hepatitis, chronic persistent hepatitis, acute hepatitis, chronic nephritis and chronic renal insufficiency, or psoriasis.

8. A detection kit containing the dimeric protein according to claim 1.

9. The detection kit according to claim 8, characterized in that the detection kit further contains an enzyme for degrading hyaluronic acid.

10. The detection kit according to claim 9, characterized in that the enzyme is hyaluronidase or its variant, a fusion protein containing hyaluronidase or its variant, or a modified hyaluronidase.

11. The detection kit according to claim 10, characterized in that the hyaluronidase is Hyal1, Hyal2, Hyal3 or mammalian testicular hyaluronidase.

12. The detection kit according to claim 11, characterized in that the hyaluronidase is human testicular hyaluronidase, and the sequence of the human testicular hyaluronidase is as shown in SEQ ID NO:

9.

13. A method for detecting hyaluronic acid for non-diagnostic purposes, characterized in that it comprises the following steps: (1) Taking a sample containing hyaluronic acid; the sample is a matrix tissue sample or a fluid sample, the matrix tissue sample is from the matrix tissue of a tumor, and the fluid sample includes blood, serum, urine, sweat, semen, saliva, cerebrospinal fluid and / or lymph sample; (2) Detecting the sample using the detection kit according to any one of claims 8 to 12.

14. The method for detecting hyaluronic acid according to claim 13, characterized in that it further comprises a step of developing color with a chromogenic solution.

15. The method for detecting hyaluronic acid according to claim 14, characterized in that the chromogenic solution is DAB.

16. The method for detecting hyaluronic acid according to any one of claims 13 to 15, characterized in that it further comprises step (3) quantitatively or semi-quantitatively analyzing the content of hyaluronic acid in the sample according to the detection result.

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