Leech thrombolytic enzyme mutant n111q, fusion protein and expression method and application

Abstract

The application provides a leech thrombolytic enzyme mutant N111Q, a fusion protein, an expression method and application, and belongs to the technical field of biological medicines. The application newly discovers a leech thrombolytic enzyme with bacteriostatic and hemolytic efficacy in leech salivary glands, and performs site-directed mutation on the leech thrombolytic enzyme, thereby obtaining the leech thrombolytic enzyme mutant N111Q. After the same prokaryotic expression as the leech thrombolytic enzyme, the activity of the obtained recombinant N111Q isopeptidase is significantly improved compared with the wild-type leech thrombolytic enzyme.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a leech thrombolytic enzyme mutant N111Q, a fusion protein, its expression method, and its applications. Background Technology

[0002] Thrombolytic enzymes are a class of protein enzymes with multiple catalytic activities, belonging to the type I lysozyme family. Their natural sources are mainly found in invertebrates, most notably medicinal leeches (including the European medicinal leech and the broad-bodied golden thread leech). The salivary glands of medicinal leeches are the main secretory sites for thrombolytic enzymes. These enzymes disrupt the host's coagulation system through thrombolytic activity, enabling them to survive by sucking blood. Thrombolytic enzymes possess both isopeptidase and glycosidase (lysozyme) activities, and also have non-enzymatic antibacterial functions, making them significant potential applications in biomedicine, the food industry, and other fields.

[0003] Currently known sources of thrombolytic enzymes are mainly the European medicinal leech and the broad-bodied golden thread leech. However, natural thrombolytic enzymes are only found in the salivary glands of leeches. The content of thrombolytic enzymes in the leech's salivary gland secretions is negligible, and the extraction process requires first separating the leech's salivary glands and then undergoing multiple purification steps. This process is cumbersome, time-consuming, and labor-intensive, making large-scale extraction difficult. Consequently, the yield of natural products cannot meet industrial and clinical needs. Furthermore, the protein function of natural thrombolytic enzymes is related to their structure and is sensitive to external conditions such as temperature, pH, and metal ions. They are prone to conformational changes at room temperature, leading to decreased enzyme activity or complete inactivation. Summary of the Invention

[0004] This invention provides a leech thrombolytic enzyme mutant N111Q, a fusion protein, an expression method, and applications. The mutant N111Q has significantly improved activity compared to wild-type leech thrombolytic enzyme, and is significantly higher than the activities of European medicinal leech thrombolytic enzyme and broad-bodied golden thread leech thrombolytic enzyme.

[0005] This invention provides a leech thrombolytic enzyme mutant, which is a point mutation occurring at position 111 of the leech thrombolytic enzyme; The amino acid sequence of the leech thrombolytic enzyme is shown in SEQ ID No. 1.

[0006] In one specific embodiment of the present invention, the point mutation is to mutate N to Q at position 111 of the amino acid sequence shown in SEQ ID No.1.

[0007] In one specific embodiment of the present invention, the amino acid sequence of the leech thrombolytic enzyme mutant is shown in SEQ ID No. 2.

[0008] The present invention also provides a nucleic acid molecule encoding the above-mentioned leech thrombolytic enzyme mutant.

[0009] In one specific embodiment of the present invention, the nucleotide sequence of the coding gene of the leech thrombolytic enzyme mutant is shown in SEQ ID No. 3.

[0010] The present invention also provides a fusion protein comprising the above-mentioned leech thrombolytic enzyme mutant, including a TEE element, a tag protein, and the leech thrombolytic enzyme mutant.

[0011] In one specific embodiment of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID No. 4.

[0012] The present invention also provides a method for expressing the above-mentioned leech thrombolytic enzyme mutant or the above-mentioned fusion protein, comprising linking the above-mentioned nucleic acid molecule or the gene encoding the fusion protein to an expression vector to construct a recombinant expression vector; The recombinant expression vector is used to transform host cells to construct engineered expression cells, and the engineered expression cells are induced to express the expression product, which contains the leech thrombolytic enzyme mutant or fusion protein.

[0013] In one specific embodiment of the present invention, the expression vector includes a prokaryotic expression vector.

[0014] The present invention also provides the use of the above-mentioned leech thrombolytic enzyme mutant, the above-mentioned fusion protein, or the leech thrombolytic enzyme mutant or fusion protein expressed by the above method in the preparation of thrombolytic and / or antibacterial drugs.

[0015] Beneficial Effects: This invention provides a leech thrombolytic enzyme mutant, which enhances the hemolytic activity of wild-type leech thrombolytic enzyme through site-directed mutagenesis. In one embodiment of this invention, the leech thrombolytic enzyme mutant and wild-type leech thrombolytic enzyme are constructed into a fusion protein using the same components, and then inserted into the NdeI-Xbale site of the pCZN1 vector to construct a prokaryotic expression system. After IPTG induction, disruption, purification, and refolding, SDS-PAGE analysis revealed an N-terminus bearing a 6-... The target protein was fused with the His tag. The thrombolytic rate of the wild-type leech thrombolytic enzyme obtained by expression was 31.59±2.43%, while the thrombolytic rate of N111Q was 36.52±0.61%. It can be seen that the thrombolytic activity of N111Q described in this invention is significantly improved compared with that of wild-type leech thrombolytic enzyme. Attached Figure Description

[0016] Figure 1The results of 12% SDS-PAGE analysis are shown in the figure. A represents wild-type leech thrombolytic enzyme HNdestabilase; B represents the leech thrombolytic enzyme mutant HNdestabilase N111Q; where M: protein molecular weight standard; 1: uninduced whole bacteria (without IPTG); 2: whole bacteria after IPTG induction; 3: sonicated supernatant; 4: sonicated precipitate; 5: supernatant after inclusion body lysis; 6: precipitate after inclusion body lysis; 7: Ni-NTA elution fraction; 8: dialysis refolded protein. Detailed Implementation

[0017] This invention provides a leech thrombolytic enzyme mutant, which is a point mutation occurring at position 111 of the leech thrombolytic enzyme; The amino acid sequence of the leech thrombolytic enzyme is shown in SEQ ID No. 1.

[0018] The thrombolytic enzyme described in this invention is derived from leeches ( Hirudo nipponia It was screened from the salivary gland transcriptome database (SRP151118) and verified to have antibacterial and hemolytic effects. Its amino acid sequence is shown in SEQ ID No.1: TVPSNCLRCICQVEGCDNEIGKCGMDMGSLSCGPYQIKEVYWIDCGRPDGDYQRCAKNKACSERCVHAYMARYALSCTGGRPPTCQDYAKIHNGGPNGCNSASNHYWDNVNRCLA.

[0019] This invention involves point mutation of the thrombolytic enzyme amino acid sequence shown in SEQ ID No. 1. The point mutation is to change N to Q at position 111 of the amino acid sequence shown in SEQ ID No. 1, named N111Q. The amino acid sequence is shown in SEQ ID No. 2: TVPSNCLRCICQVEGCDNEIGKCGMDMGSLSCGPYQIKEVYWIDCGRPDGDYQRCAKNKACSERCVHAYMARYALSCTGGRPPTCQDYAKIHNGGPNGCNSASNHYWDNVQRCLA.

[0020] The present invention also provides a nucleic acid molecule encoding the above-mentioned leech thrombolytic enzyme mutant.

[0021] This invention expresses the mutant N111Q. In one embodiment, the mutant N111Q is expressed using a prokaryotic expression method. The nucleotide sequence of the coding gene for the leech thrombolytic enzyme mutant is shown in SEQ ID No. 3: ACAGTACCCTCAAATTGTCTAAGGTGTATCTGCCAGGTTGAAGGTTGCGACAACGAGATCGGCAAGTGCGGCATGGACATGGGTTCGTTGTCTTGTGGTCCGTATCAGATTAAAGAAGtgTACTGGATTGACTGTGGCCGTCCGGATGGTGACTACCAACGTTGCGCTAAGAACAAAGCGTGTTCCGAGCGCTGCGTGCACGCTTATATGGCGCGTTATGCGCTGAGCTGTACCGGTGGTCGCCCACCGACGTGCCAAGATTACGCCAAGATCCACAACGGCGGTCCGAATGGCTGCAACAGCGCAAGCAATCATTACTGGGATAACGTCCAGCGTTGCCTGGCGTAA.

[0022] The present invention also provides a fusion protein comprising the above-mentioned leech thrombolytic enzyme mutant, including a TEE element, a tag protein, and the leech thrombolytic enzyme mutant.

[0023] The TEE element described in this invention can promote the translation of subsequent proteins. In one embodiment, the amino acid sequence of the TEE element is shown in SEQ ID No. 5: MNHKV; the tag protein used for prokaryotic expression is His. The 6-tag designation is used to construct the N111Q mutant fusion protein, whose amino acid sequence is shown in SEQ ID No. 4: MNHKVHHHHHHMTVPSNCLRCICQVEGCDNEIGKCGMDMGSLSCGPYQIKEVYWIDCGRPDGDYQRCAKNKACSERCVHAYMARYALSCTGGRPPTCQDYAKIHNGGPNGCNSASNHYWDNVQRCLA. The nucleotide sequence of the encoding gene is shown in SEQ ID No. 4. Shown in No.5: ATGAACCATAAGGTTCATCATCATCATCATATGACAGTACCCTCAAATTGTCTAAGGTGTATCTGCCAGGTTGAAGGTTGCGACAACGAGATCGGCAAGTGCGGCATGGACATGGGTTCGTTGTCTTGTGGTCCGTATCAGATTAAAGAAGtgTACTGGATTGACTGTGGCCGTCCGGATGGTGAC TACCAACGTTGCGCTAAGAACAAAGCGTGTTCCGAGCGCTGCGTGCACGCTTATATGGCGCGTTATGCGCTGAGCTGTACCGGTGGTCGCCCACCGACGTGCCAAGATTACGCCAAGATCCACAACGGCGGTCCGAATGGCTGCAACAGCGCAAGCAATCATTACTGGGATAACGTCCAGCGTTGCCTGGCGTAA.

[0024] In one embodiment of the present invention, the same method was used to express wild-type leech thrombolytic enzyme in prokaryotes, thus constructing a prokaryotes expression system with the amino acid sequence shown in SEQ ID No. 6: MNHKVHHHHHHMTVPSNCLRCICQVEGCDNEIGKCGMDMGSLSCGPYQIKEVYWIDCGRPDGDYQRCAKNKACSERCVHAYMARYALSCTGGRPPTCQDYAKIHNGGPNGCNSASNHYWDNVNRCLA. The nucleotide sequence of the encoding gene is shown in SEQ ID No. 6. Shown in No.7: ATGAACCATAAGGTTCATCATCATCATCATATGACCGTTCCTAGTAATTGTCTGCGCTGCATTTGTCAGGTTGAAGGCTGCGATAATGAAATTGGTAAATGTGGTATGGACATGGGCAGTCTGAGCTGCGGCCCTTATCAGATTAAGGAAGTTTATTGGATCGACTGTGGTCGTCCGGATGGTGAC TATCAGCGCTGCGCAAAAAATAAGGCATGTAGTGAACGTTGCGTTCATGCCTATATGGCACGCTATGCCCTGAGCTGCACCGGTGGTAGACCGCCTACATGCCAGGATTATGCCAAAATTCATAACGGTGGCCCGAATGGTTGCAATAGCGCCAGTAATCATTATTGGGATAACGTTAACCGCTGCCTGGCCTAA.

[0025] The present invention also provides a method for expressing the above-mentioned leech thrombolytic enzyme mutant or the above-mentioned fusion protein, comprising linking the above-mentioned nucleic acid molecule or the gene encoding the fusion protein to an expression vector to construct a recombinant expression vector; The recombinant expression vector is used to transform host cells to construct engineered expression cells, and the engineered expression cells are induced to express the expression product, which contains the leech thrombolytic enzyme mutant or fusion protein.

[0026] The present invention does not specifically limit the expression method, and can use prokaryotic expression or eukaryotic expression. When using prokaryotic expression, Escherichia coli can be used as the expression host; when using eukaryotic expression, the expression host can be yeast, plant, insect or animal.

[0027] In one embodiment of the present invention, a prokaryotic expression method is used for the expression. Therefore, a prokaryotic expression vector is used during construction, including inserting the sequence shown in SEQ ID No. 5 or the sequence shown in SEQ ID No. 7 into the NdeI-Xbale site of the pCZN1 vector, and then transforming the recombinant expression vector into E. coli (BL21) competent bacteria to construct the Hndestabilase prokaryotic expression system, and inducing expression based on the expression mode of inclusion bodies, wherein the induced expression includes IPTG induced expression.

[0028] The present invention also provides the use of the above-mentioned leech thrombolytic enzyme mutant, the above-mentioned fusion protein, or the leech thrombolytic enzyme mutant or fusion protein expressed by the above method in the preparation of thrombolytic and / or antibacterial drugs.

[0029] In this embodiment of the invention, the above-mentioned induced expression system, after purification and refolding steps following induced expression, was analyzed by SDS-PAGE to obtain N-terminus 6- ... The target protein was fused with a His tag. The antibacterial activity of the wild-type leech thrombolytic enzyme obtained by expression was 28.90±1.43 μg / mgprot, and the thrombolysis rate of old thrombi was 31.59±2.43%, while the antibacterial activity of N111Q was 28.50±1.53 μg / mgprot, and the thrombolysis rate of old thrombi was 36.52±0.61%. It can be seen that compared with the wild-type leech thrombolytic enzyme, the N111Q described in this invention has significantly improved activity in dissolving old thrombi.

[0030] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a leech thrombolytic enzyme mutant N111Q, a fusion protein, its expression method, and its applications, should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 1. Construction, induction, and purification of fusion expression systems A fusion protein was constructed using leech thrombolytic enzyme, with the encoding gene shown in SEQ ID No. 7. A similar fusion protein was constructed using the mutant N111Q, with the encoding gene shown in SEQ ID No. 5. Based on the sequences of the broad-bodied golden thread leech thrombolytic enzyme fusion protein and the enhanced-activity broad-bodied golden thread leech thrombolytic enzyme fusion protein disclosed in Chinese patents CN119080950A and CN119241723A, the same fusion expression system, induced expression, purification, and subsequent experiments were performed. The same methods were used for subsequent protein activity detection for comparison. Furthermore, the deletion of 10 amino acids at the C-terminus of the original broad-bodied golden thread leech thrombolytic enzyme “pCZN1-Wpdestabilase”, i.e., “pCZN1-Wpdestabilase-without” = “pCZN1-WpDestabilase C10”, was verified. The same experiment was also conducted on *Hirudo medicinalis*, and the relevant nucleotide sequence of the *Hirudo medicinalis* thrombolytic enzyme fusion protein is shown in SEQ ID No. 10: ATGAACCATAAGGTTCATCATCATCATCATCATATGACTGTACCGTCCGACTGTCTGCGTTGCATCTGCCAGGTTGAAGGCTGTAACAACGAAATCGGTCGTTGCGGTATGGATGCGGGCTCTCTCAGCTGCGGTCCATACCAGATCAAAGAACCGTACTGGATCGACTGTGGTCGTCCGGGTGGTGGTTACCAGCAGTGCACCAAAGAGAAAGCGTGCTCCGAACGTTGTGTTCACGCTTACATGGATCGCTACGCACGTCGTTGCACTGGTGGTCGCCAGCCGACCTGCCAAGATTACGCGAAGATCCACAACATGGGTCCAAACTGCTGCCAATCTTCTAACAACCACTACTGGGACAACGTTCGTCGTTGCCTGGGTTAA.

[0032] The sequences shown in SEQ ID No. 5 and SEQ ID No. 7 were inserted into the NdeI-Xbale site of the pCZN1 vector, and then the recombinant expression vector was transformed into E. coli (BL21) competent bacteria to construct the Hndestabilase prokaryotic expression system.

[0033] Activate E. coli containing the Hndestabilase recombinant plasmid on LB agar plates. Selected single colonies are inoculated into 3 mL LB medium containing 50 μg / mL Amp and incubated overnight at 37°C with shaking at 200 rpm. The next day, inoculate 1:100 into 100 mL LB medium containing 50 μg / mL Amp and incubate at 37°C with shaking at 200 rpm until the bacterial cell OD reaches 100%. 600 The concentration was 0.6–0.8. Take 1 mL of culture, centrifuge at 10,000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet in 100 μL of 1× loading buffer. Add Isopropyl-β-D-thiogalactopyranoside (IPTG) to the remaining culture to a final concentration of 0.2 mM, and incubate overnight at 15°C with shaking at 200 r / min to induce fusion protein expression. Take 1 mL of culture, centrifuge at 10,000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet in 100 μL of 1× loading buffer. Centrifuge the remaining culture at 4000 r / min for 10 min, discard the supernatant, and resuspend the bacterial pellet in PBS; after sonication of the resuspended solution, take the supernatant and pellet separately and resuspend them in loading buffer. Perform 12% SDS-PAGE analysis, and stain with Coomassie Brilliant Blue for banding. The total amount of recombinant N111Q target protein obtained after purification and dialysis was 3.4 mg, with a concentration of 0.425 mg / mL; the amount of wild-type recombinant target protein was 3 mg, with a concentration of 0.313 mg / mL.

[0034] The inclusion body protein refolding procedure is as follows: The bacterial cell pellet was resuspended in 20 mL of lysis buffer (20 mM Tris-HCl containing 1 mM PMSF and a bacteria protease inhibitor cocktail, pH 8.0), and sonicated (400 W, 4 sec on, 8 sec on, 20 min total). The sonicated cell lysate was centrifuged at 10,000 r / min for 20 min at 4°C, and the pellet was collected. The inclusion bodies were washed three times with inclusion body washing buffer (20 mM Tris, 1 mM EDTA, 2 M urea, 1 M NaCl, 1% Triton X-100, pH 8.0). The inclusion bodies were dissolved in lysis buffer (20 mM Tris, 5 mM DTT, 0.15 M NaCl, 8 M urea, pH 8.0), sonicated (400 W, 4 sec on, 8 sec on, 15 min total), and centrifuged at 10,000 r / min for 15 min at room temperature. The supernatant was collected.

[0035] The refolded fusion protein was purified by Ni-column affinity chromatography using a low-pressure chromatography system. The supernatant was loaded onto a Ni-IDA-Sepharose Cl-6B affinity chromatography column pre-equilibrated with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min. The column was washed with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min until the effluent reached OD. 280 The value reached baseline. Wash with Ni-IDA Washing-Buffer (20 mM Tris-HCl, 20 mM imidazole, 0.15 M NaCl, 8 M urea, pH 8.0) at a flow rate of 1 mL / min until the OD of the outflow reached baseline. 280 Values ​​reached baseline. The target protein was eluted with Ni-IDA Elution-Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, 8 M urea, pH 8.0) at a flow rate of 1 mL / min, and the eluent was collected. The collected protein solution was added to a dialysis bag and dialyzed into refolding buffer overnight. After refolding, it was dialyzed into PBS for storage. 12% SDS-PAGE analysis was performed. The concentration of hndestabilase was determined using a BCA protein assay kit (Sangon Biotech, China).

[0036] SDS-PAGE analysis results are as follows: Figure 1 As shown, the fusion protein of this invention is an inclusion body protein that can be induced to express in E. coli. After processes such as refolding and purification, a soluble protein is finally obtained.

[0037] 2. Detection of in vitro thrombolytic activity Fresh pig blood was placed in a test tube, and whole blood was mixed with 3.8% sodium citrate anticoagulant at a ratio of 9:1. Then, 20 μL of 0.5% fibrinogen solution, 10 μL of 0.5 mol / L CaCl2 solution, and 20 μL of 100 U / ml thrombin solution were added sequentially to each 1 mL of blood. After rapid mixing, the blood was injected into a polyethylene tube with an inner diameter of 8 mm. After incubating at 37 ℃ for 1 h, the fresh blood clot was removed. The clot was rinsed with physiological saline until the liquid was colorless, then placed in a 37 ℃ incubator for 1 h. The clot was then removed, turned over, and placed back into the 37 ℃ incubator for 1 h to obtain a fresh thrombus. Fresh thrombi stored at 4 ℃ for 48 h are considered old thrombi.

[0038] Add 0.1 g of fresh or old thrombus to 1 mL of test sample and place in a 37℃ constant temperature shaker at 60 r / min for 48 h. Remove the remaining thrombus, rinse the surface of the blood clot with physiological saline to remove impurities, place in an oven at 37℃ for 1 h, turn the blood clot over, and then treat it again at 37℃ for 1 h. Weigh the clot and calculate the thrombolysis rate according to the formula. Each group was repeated 3 times.

[0039] Thrombolysis rate = [(mass of thrombus before dissolution - mass of thrombus after dissolution) / mass of thrombus before dissolution] × 100%.

[0040] The results are shown in Table 1. In terms of the thrombolysis rate of fresh thrombi, the leech thrombolytic enzyme fusion protein (pCZN1-Hndestabilase) was 41.70±0.28%, the European medicinal leech thrombolytic enzyme (pCZN1-Hmdestabilase) was 41.18±1.30%, the broad-bodied golden thread leech thrombolytic enzyme (pCZN1-Wpdestabilase) was 55.16±3.85%, while the N111Q fusion protein (pCZN1-Hndestabilase N111Q) of this application can reach 46.78±1.67%, which is significantly improved in activity compared with natural leech thrombolytic enzyme. In terms of the thrombolysis rate of old thrombi, the leech thrombolytic enzyme fusion protein (pCZN1-Hndestabilase) was 31.59±2.43%, the European medical leech thrombolytic enzyme (pCZN1-Hmdestabilase) was 31.52±1.36%, the broad-bodied golden thread leech thrombolytic enzyme (pCZN1-Wpdestabilase) was 36.18±2.16%, while the N111Q fusion protein (pCZN1-HndestabilaseN111Q) of this application can reach 36.52±0.61%, which is also significantly improved in activity compared with natural leech thrombolytic enzyme.

[0041] Table 1. Activity assay results of the fusion protein

[0042] 3. Detection of isopeptidase activity The isopeptidase activity assay is based on the principle that isopeptidase cleaves L-γ-glutamine-p-nitroanilide (L-γ-Glu-pNA; Sigma) to generate p-nitroaniline (pNA). The absorbance of pNA at 405 nm before and after the reaction was measured, and the difference in absorbance before and after the reaction was taken as the isopeptidase activity of the sample. A 0.25 mg / mL L-γ-Glu-pNA substrate solution was prepared using 50 mM 3-morpholinopropanesulfonic acid buffer (pH 7.0) containing 10 mM NaCl. 50 μL of the sample was mixed with 100 μL of the substrate and incubated at 37 °C for 24 h. The absorbance at 405 nm before and after the reaction was measured, with PBS (without the sample) used as a control. The assay was repeated three times.

[0043] The results are shown in Table 1. The thrombolytic enzyme fusion protein of leeches (pCZN1-Hndestabilase) was 0.331±0.062, that of European medicinal leech thrombolytic enzyme (pCZN1-Hmdestabilase) was 0.450±0.005%, that of broad-bodied golden leech thrombolytic enzyme (pCZN1-Wpdestabilase) was 0.415±0.052, while the N111Q fusion protein of this application (pCZN1-Hndestabilase N111Q) can reach 0.425±0.011.

[0044] 4. Detection of lysozyme activity The lysozyme activity of the samples was detected using a self-control method, following the instructions of the lysozyme assay kit (Nanjing Jiancheng Bioengineering Institute). The specific steps are as follows: 2 mL of the applied bacterial suspension (Micrococcus luteus) was rapidly added to a cuvette containing 0.2 mL of the test sample. After thorough mixing, the transmittance at 530 nm was measured at 15 s and 2 min 15 s, respectively, and recorded as T0 and T1. The transmittance changes after mixing the standard and the applied bacterial suspension were also measured at 15 s and 2 min 15 s, respectively, and recorded as T2 and T3. Each sample was tested three times in duplicate. The lysozyme content of the sample was calculated using the formula: Lysozyme content (μg / mgprot) = (sample ΔT ÷ standard ΔT) × standard concentration ÷ sample protein concentration (μg / mL), where sample ΔT = T1 - T0, and standard ΔT = T3 - T2.

[0045] The results are shown in Table 1. The thrombolytic enzyme fusion protein of leeches (pCZN1-Hndestabilase) was 28.90±1.43 μg / mgprot, that of European medicinal thrombolytic enzyme (pCZN1-Hmdestabilase) was 22.53±1.92 μg / mgprot, that of broad-bodied golden thread thrombolytic enzyme (pCZN1-Wpdestabilase) was 35.64±3.27 μg / mgprot, and that of the N111Q fusion protein of this application (pCZN1-Hndestabilase N111Q) was 28.50±1.53 μg / mgprot.

[0046] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A leech thrombolytic enzyme mutant, characterized in that, This is a point mutation occurring at position 111 of the leech thrombolytic enzyme; The amino acid sequence of the leech thrombolytic enzyme is shown in SEQ ID No.

1.

2. The leech thrombolytic enzyme mutant according to claim 1, characterized in that, The point mutation is to change N to Q at position 111 of the amino acid sequence shown in SEQ ID No.

1.

3. The leech thrombolytic enzyme mutant according to claim 1 or 2, characterized in that, The amino acid sequence of the leech thrombolytic enzyme mutant is shown in SEQ ID No.

2.

4. A nucleic acid molecule encoding a leech thrombolytic enzyme mutant according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleotide sequence of the gene encoding the leech thrombolytic enzyme mutant is shown in SEQ ID No.

3.

6. A fusion protein comprising the leech thrombolytic enzyme mutant according to any one of claims 1 to 3, characterized in that, This includes TEE elements, tag proteins, and leech thrombolytic enzyme mutants.

7. The fusion protein according to claim 6, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID No.

4.

8. A method for expressing the leech thrombolytic enzyme mutant according to any one of claims 1 to 3 or the fusion protein according to claim 6 or 7, characterized in that, This includes connecting the nucleic acid molecule of claim 4 or 5 or the gene encoding the fusion protein to an expression vector to construct a recombinant expression vector; The recombinant expression vector is used to transform host cells to construct engineered expression cells, and the engineered expression cells are induced to express the expression product, which contains the leech thrombolytic enzyme mutant or fusion protein.

9. The method according to claim 8, characterized in that, The expression vectors include prokaryotic expression vectors.

10. The use of the leech thrombolytic enzyme mutant according to any one of claims 1 to 3, the fusion protein according to claim 6 or 7, or the leech thrombolytic enzyme mutant or fusion protein expressed by the method according to claim 8 or 9 in the preparation of thrombolytic and / or antibacterial drugs.

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