Whitmania pigra destabilizing enzyme fusion protein and application thereof

Abstract

The invention discloses a destabilizing enzyme fusion protein with improved activity, and the amino acid sequence of the destabilizing enzyme fusion protein is as shown in SEQ ID No.3 or SEQ ID No.5. According to the destabilizing enzyme fusion protein disclosed by the invention, the bacteriostatic activity, thrombolytic activity and isopeptidase activity of the destabilizing enzyme fusion protein are improved by optimizing the expression strategy and modifying the sequence of destabilizing enzyme Wpdestalase from whitmania pigra; and the application prospect and the application space of the Wpestabase are improved.

Description

[0001] The present invention is a divisional application of Chinese patent application CN 2024113424937, whose application date is September 25, 2024, and whose name is “A wide-bodied golden thread leech destabilizing enzyme fusion protein and its application”. Technical Field

[0002] The invention relates to the field of biotechnology, and in particular to an instabilizing enzyme fusion protein derived from a broad-bodied golden leech and an application thereof. Background Art

[0003] The wide-bodied golden leech (Whitmania pigra Whitman) belongs to the order Hirudinidae, family Hirudinidae, genus Whitmania. It is one of the basal animals of the traditional Chinese medicinal leech and has great medicinal value. It is widely distributed in my country and lives in paddy fields and ditches all year round, feeding on fresh snail body fluids.

[0004] The applicant screened a leech polypeptide transcript with antibacterial and hemolytic effects from the W.pigra salivary gland transcriptome database (SRX16840326, SRX16840327, SRX16840328), and verified the correctness of the sequence by cDNA sequence cloning. After removing the polypeptide signal peptide, the full-length splicing primer was designed based on the PAS method, the target gene was synthesized, and the target gene was inserted into the NcoⅠ and XhoⅠ restriction sites of the pET-32a vector to construct the pET32a-Trx-EK-wpDestabilase prokaryotic expression system. After IPTG induction, fragmentation, purification, and SDS-PAGE detection, a fusion target protein with Trx and other tags at the N-terminus was obtained. The antibacterial activity of the fusion target protein was 14.17 μg / mL, and the hemolysis rate was 44.06%; for details, see the Chinese invention patent, application number: CN202111590989.2; authorization announcement number: CN114057866B.

[0005] In order to further improve the activity of the destabilizing enzyme (WpDestabilase) of Hirudo spp., the applicant optimized the expression strategy of WpDestabilase and obtained a fusion protein with significantly improved antibacterial and thrombolytic activities. Summary of the invention

[0006] In one aspect, the present invention provides a destabilizing enzyme fusion protein with enhanced activity, wherein the amino acid sequence of the destabilizing enzyme fusion protein is shown in SEQ ID No.3 or SEQ ID No.5.

[0007] In one embodiment, the amino acid sequence of the destabilizing enzyme fusion protein is shown as SEQ ID No.3.

[0008] In one embodiment, the amino acid sequence of the destabilizing enzyme fusion protein is shown as SEQ ID No.5.

[0009] On the other hand, the present invention also provides a gene encoding the above-mentioned destabilizing enzyme fusion protein.

[0010] In one embodiment, the sequence of the gene encoding the destabilizing enzyme fusion protein is shown as SEQ ID No.4.

[0011] In one embodiment, the sequence of the gene encoding the destabilizing enzyme fusion protein is shown as SEQ ID No.6.

[0012] On the other hand, the present invention also provides a biological material comprising the above destabilizing enzyme fusion protein or its encoding gene. The biological material is selected from: a vector comprising the above destabilizing enzyme fusion protein or its encoding gene, or a host cell comprising the above destabilizing enzyme fusion protein or its encoding gene.

[0013] On the other hand, the present invention also provides a vector comprising the above encoding gene, or a host cell comprising the vector.

[0014] In one embodiment, the vector includes a cloning vector and an expression vector.

[0015] In one embodiment, the vector is pCZN1.

[0016] In one embodiment, the vector is circular or linear.

[0017] As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different host cells. An "expression vector" refers to a vector that has the ability to incorporate and express heterologous DNA fragments in exogenous cells. Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of an appropriate expression vector is well known to those skilled in the art.

[0018] In one embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0019] In one embodiment, the prokaryotic cell is derived from Escherichia coli or Agrobacterium.

[0020] In one embodiment, the eukaryotic cell is yeast.

[0021] On the other hand, the present invention also provides the use of the above-mentioned destabilizing enzyme fusion protein, or a biomaterial comprising the above-mentioned destabilizing enzyme fusion protein or a gene encoding the same, in thrombus dissolution and / or bacteriostasis.

[0022] On the other hand, the present invention also provides the use of the above-mentioned destabilizing enzyme fusion protein, or a biological material comprising the above-mentioned destabilizing enzyme fusion protein or a gene encoding the same, in the preparation of an agent for dissolving thrombus and / or inhibiting bacteria.

[0023] In one embodiment, the bacteriostasis is inhibition of cocci, for example, Micrococcus luteus.

[0024] In one embodiment, the thrombus is selected from one or any combination of fresh thrombus and aged thrombus (or also referred to as old thrombus).

[0025] On the other hand, the present invention also provides a method for preparing the above-mentioned destabilizing enzyme fusion protein, which comprises the step of expressing the above-mentioned destabilizing enzyme fusion protein using an expression vector.

[0026] Furthermore, the method also includes the step of separating / purifying the destabilizing enzyme fusion protein.

[0027] The invention improves the antibacterial activity, thrombolytic activity and isopeptidase activity of the destabilase fusion protein by optimizing the expression strategy of the destabilase WpDestabilase from the wide-bodied golden line leech, thereby improving the application prospect and application space of WpDestabilase. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Expression, purification and renaturation results of pCZN1-WpDestabilase fusion protein; wherein, a, expression of pCZN1-WpDestabilase fusion protein. M: protein marker, lane 1: CK (whole cell lysate after induction with pCZN1 empty vector), lane 2: CK (without IPTG), lane 3: after induction, lane 4: supernatant after induction and disruption, lane 5: precipitate after induction and disruption. b. purification of pCZN1-WpDestabilase fusion protein. M: protein marker, lane 1: precipitate after induction and disruption of pCZN1-WpDestabilase fusion protein. Lane 2: circulating liquid. Lanes 3, 4: protein obtained by elution. c, protein detection analysis after renaturation of pCZN1-WpDestabilase fusion protein. M: protein marker, lane 1: detection results after renaturation and purification of pCZN1-WpDestabilase fusion protein.

[0029] Figure 2: Expression, purification and renaturation results of pCZN1-WpDestabilaseC10 fusion protein; wherein, a, expression of pCZN1-WpDestabilaseC10 fusion protein. M: protein marker, lane 1: CK (whole cell lysate after induction with pCZN1 empty vector), lane 2: CK (without IPTG), lane 3: after induction, lane 4: supernatant after induction and disruption, lane 5: precipitate after induction and disruption. b. purification of pCZN1-WpDestabilaseC10 fusion protein. M: protein marker, lane 1: precipitate after induction and disruption of pCZN1-WpDestabilaseC10. Lane 2: flowing liquid. Lanes 3, 4: protein obtained by elution. c, protein detection analysis after renaturation of pCZN1-WpDestabilaseC10 fusion protein. M: protein marker, lane 1: detection results after renaturation and purification of pCZN1-WpDestabilaseC10 fusion protein. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the examples. The following description is only a preferred embodiment of the present invention and is not intended to limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. The materials, reagents, instruments and methods used in the following examples are conventional materials, reagents, instruments and methods in the art unless otherwise specified, and can be obtained through commercial channels.

[0031] Any simple modification or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention shall fall within the protection scope of the present invention.

[0032] Example 1. Expression, purification and renaturation of pCZN1-WpDestabilase fusion protein

[0033] As described in the aforementioned patent (Chinese invention patent application, application number: CN202111590989.2), the amino acid sequence of the destabilizing enzyme WpDestabilase from Hirudo sphaerocephala is as follows (SEQ ID No. 1):

[0034] MMKSAIYSCFALLTIVLALSEVNS QISDPCLRCICKEEGCETQIGQCNDGTSQSCGPYQ IMRAYWIDCGKPGNDYETCTKTIDCSEACVRAYMNRYGTYCTGGRTPTCQDYARIHKGGPSGCNQSETFVYGKKVQECSVIPATETTTEI (the underlined part is the signal peptide).

[0035] The amino acid sequence of the pET32a-Trx-EK-WpDestabilase fusion target protein used in the above patent is as follows (SEQ ID No. 2):

[0036] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQ HMDSPDLGTDDDDKAMASQISDPCLRCICKEEGCETQIGQCNDGTSQSCGPYQIMRAYWIDCGKPGNDYETCTKTIDCSEACVRAYMNRYGTYCTGGRTPTCQDYARIHKGGPSGCNQSETFVYGKKVQECSVIPATETTTEI.

[0037] The N-terminus of the pET32a-Trx-EK-wpDestabilase protein is fused with the TrxA, His, thrombin recognition site, S-tag tag and enterokinase (EK) recognition site sequences of the pET32a vector.

[0038] In this example, in order to improve the activity of the WpDestabilase fusion protein, an attempt was made to change the expression strategy and reduce the amino acid sequence of non-target polypeptides.

[0039] In this embodiment, the pCZN1 expression vector was used to express WpDestabilase to reduce the number of redundant amino acid sequences other than the target polypeptide. The amino acid sequence of the obtained fusion protein pCZN1-WpDestabilase is shown below (SEQ ID No. 3), and its gene sequence is shown in SEQ ID No. 4:

[0040] MNHKVHHHHHHMQISDPCLRCICKEEGCETQIGQCNDGTSQSCGPYQIMRAYWIDC GKPGNDYETCTKTIDCSEACVRAYMNRYGTYCTGGRTPTCQDYARIHKGGPSGCNQSET FVYGKKVQECSVIPATETTTEI (SEQ ID No. 3).

[0041] ATGAATCACAAAGTGCATCATCATCATCATATGCAGATCAGTGATCCGTGTCTGCGCTGTATTTGTAAAGAAGAAGGTTGTGAAACCCAGATTGGTCAGTGCAATGATGGCACCAGTCAGAGTTGTGGTCCGTATCAGATTATGCGTGCATATTGGATTGACTGTGGCAAACCGGGTAATGATTATGAAACCTGCACCAAAACCAT CGATTGTAGTGAAGCATGTGTGCGTGCATATATGAATCGTTATGGCACCTATTGTACCGGCGGTCGTACCCCTACCTGTCAGGATTATGCCCGTATTCATAAAGGTGGTCCGAGTGGTTGTAATCAGAGCGAAACCTTTGTTTATGGCAAAAAAGTGCAGGAATGCAGCGTGATTCCGGCAACCGAAACCACCACCGAAATTTAA(SEQ ID No.4).

[0042] In order to further change the amino acid sequence of the target polypeptide, the 10aa amino acid sequence "IPATETTTEI" at the C-terminus of the WpDestabilase target polypeptide was also deleted in this embodiment, and the sequence was recorded as pCZN1-WpDestabilaseC10, whose amino acid sequence is shown as follows (SEQ ID No.5), and its gene sequence is shown as SEQ ID No.6:

[0043] MNHKVHHHHHHMQISDPCLRCICKEEGCETQIGQCNDGTSQSCGPYQIMRAYWIDC GKPGNDYETCTKTIDCSEACVRAYMNRYGTYCTGGRTPTCQDYARIHKGGPSGCNQSET FVYGKKVQECSV (SEQ ID No. 5).

[0044] ATGAATCACAAAGTGCATCATCATCATCATCATATGCAGATCAGCGATCCGTGTCTGCGTTGCATTTGCAAAGAAGAAGGCTGCGAAACCCAGATTGGTCAGTGCAATGATGGTACCAGTCAGAGTTGCGGTCCGTATCAGATTATGCGCGCATATTGGATTGATTGTGGCAAACCGGGTAATGATTATGAAACCTGTACCAAAACCATCGATTGCAGCGAAGCCTGTGTGCGTGCATATATGAATCGCTATGGTACCTATTGCACCGGTGGTCGTACCCCGACCTGTCAAGATTATGCCCGCATTCATAAAGGCGGTCCGAGTGGTTGTAATCAGAGTGAAACCTTTGTGTATGGCAAAAAAGTGCAGGAATGCAGCGTTTAA(SEQ ID No.6)。

[0045] Specifically: In this embodiment, after removing the target polypeptide signal peptide sequence from the destabilizing enzyme WpDestabilase from the wide-bodied golden line leech, the target gene sequence was artificially synthesized and connected to the pCZN1 plasmid to construct the pCZN1-WpDestabilase recombinant plasmid. The qualified recombinant plasmid was introduced into the competent bacteria of Escherichia coli (Arctic-Express) to construct the WpDestabilase prokaryotic expression system. The Escherichia coli containing the WpDestabilase recombinant plasmid was activated on the LB plate, and the picked single clone was inoculated in a test tube containing 3mL LB culture medium containing 50μg / mL Amp, and shaken at 37℃200r / min overnight. The next day, it was inoculated in 100mL LB culture medium containing 50μg / mL Amp at 1:100, and shaken at 37℃200r / min until the bacterial OD600 was 0.6-0.8. Take out 1mL of culture, centrifuge at 10000r / min at room temperature for 2min, discard the supernatant, and resuspend the bacterial pellet with 100μL 1× loading buffer. Add Isopropyl-β-D-thiogalactopyranoside (IPTG) to the remaining culture to a final concentration of 0.2mM, shake at 15℃ 200r / min overnight to induce fusion protein expression. Take out 1mL of culture, centrifuge at 10000r / min at room temperature for 2min, discard the supernatant, and resuspend the bacterial pellet with 100μL 1× loading buffer. Centrifuge the remaining culture at 4000r / min for 10min, discard the supernatant, and resuspend the bacterial pellet with PBS; after ultrasonically disrupting the resuspension, take the supernatant and the precipitate respectively, add the loading buffer and resuspend. Perform 12% SDS-PAGE detection and analysis, and Coomassie Brilliant Blue staining to show bands.

[0046] The steps for inclusion body protein refolding are as follows: resuspend the bacterial pellet in 20mL lysis buffer (20mM Tris-HClcontaining1mM PMSF and bacteria protease inhibitor cocktail, pH 8.0), and ultrasonically disrupt (power 400W, working 4sec, intermittent 8sec, total 20min). Centrifuge the ultrasonically disrupted cell lysate at 4℃10000r / min for 20min, and collect the precipitate. Wash the inclusion bodies 3 times with inclusion body washing buffer (20mM Tris, 1mM EDTA, 2M urea, 1M NaCl, 1% TritonX-100, pH 8.0). Dissolve the inclusion bodies with lysis buffer (20mM Tris, 5mM DTT, 0.15M Nacl, 8M urea, pH 8.0), ultrasonically disrupt (power 400W, working 4sec, intermittent 8sec, total 15min), and centrifuge at room temperature, 10000r / min for 15min. Collect the supernatant.

[0047] The steps of refolded fusion protein Ni column affinity purification are as follows: using a low-pressure chromatography system, the supernatant solution is 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. Rinse with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min until the OD280 value of the effluent reaches the baseline. Rinse with Ni-IDAWashing-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 OD280 value of the effluent reaches the baseline. Elute the target protein 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 collect the effluent. The collected protein solution was added to a dialysis bag, dialyzed into a refolding buffer overnight, and after the refolding was completed, dialyzed into PBS for storage. 12% SDS-PAGE analysis was performed.

[0048] The expression, purification, and renaturation results of the pCZN1-WpDestabilase fusion protein are shown in Figure 1 The expression, purification and renaturation results of pCZN1-WpdestabilaseC10 fusion protein are shown in Figure 2 shown.

[0049] Example 2: Verification of WpDestabilase fusion protein activity

[0050] In this embodiment, the thrombolytic activity, isopeptidase activity, fibrinolytic activity and lysozyme activity of the pCZN1-WpDestabilase and pCZN1-WpDestabilaseC10 fusion proteins obtained in Example 1 were detected, and aspirin, PBS and the Chinese invention patent application CN202111590989.2 were also detected.

[0051] Activity of pET32a-Trx-EK-WpDestabilase fusion protein.

[0052] 2.1 Detection of in vitro thrombolytic activity

[0053] Place fresh pig blood in a test tube, add 3.8% anticoagulant sodium citrate to the whole blood at a ratio of 9:1, then add 20μL of 0.5% fibrinogen solution, 10μL of 0.5mol / L CaCl2 solution, and 20μL of 100U / ml thrombin solution to each mL of blood, mix quickly, and inject the blood into a polyethylene tube with an inner diameter of 8mm. After 1 hour in a 37℃ water bath, take out the fresh blood clot. Rinse with physiological saline until the liquid is colorless, place it in a 37℃ incubator, bake it for 1 hour, take out the blood clot, turn it over, and put it back in the 37℃ incubator. After baking for 1 hour, you will get fresh thrombus. Fresh thrombus stored at 4℃ for 48 hours is considered old thrombus (Wyshelesky et al., 2001).

[0054] 0.1g of fresh thrombus or old thrombus was added to 1mL of test sample, and the sample was placed in a 37℃ constant temperature shaker and shaken slowly at 60r / min for 48h. The remaining thrombus was removed, and the impurities on the surface of the blood clot were rinsed with physiological saline, and the blood clot was placed in an oven at 37℃ for 1h. After the blood clot was turned over and treated at 37℃ for another 1h, it was weighed and the thrombolysis rate was calculated according to the following formula. Each group was repeated 3 times. Thrombolysis rate = [(thrombus mass before lysis - thrombus mass after lysis) / thrombus mass before lysis] × 100%.

[0055] 2.2 Detection of isopeptidase activity

[0056] 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 is measured, and the difference in absorbance before and after the reaction is calculated as the isopeptidase activity of the test sample (Bathige et al., 2013). 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 test sample was mixed with 100 μL of the substrate and incubated at 37°C for 24 h. The absorbance at 405 nm was measured before and after the reaction, and PBS without the test sample was used as a control. The test was repeated three times.

[0057] 2.3 Detection of fibrin plate lysis

[0058] After mixing 20mL of 1% agarose solution and 8mL of 0.5% fibrinogen solution, add 1mL of 10U / mL thrombin solution, stir evenly, and immediately pour into a sterilized culture dish (9cm in diameter), shake gently, and let it stand and cool for about 30min until the agar is completely solidified to make a fibrin plate. Punch holes (8mm in diameter) on the fibrin plate, with a hole distance greater than 1.5cm on the plate, add 5μL of the test sample into the hole, bake in a 37℃ oven overnight, observe the dissolution zone, and repeat 5 times.

[0059] 2.4 Detection of lysozyme activity

[0060] The lysozyme activity of the sample was detected by the self-control method according to the operating steps of the lysozyme assay kit manual (Nanjing Jiancheng Bioengineering Institute). The specific steps are as follows. 2 mL of the applied bacterial solution (Micrococcus luteus) was quickly injected into a cuvette containing 0.2 mL of the test sample, mixed evenly, and then the transmittance value at 530 nm was detected at 15 s and 2 min 15 s, respectively, recorded as T0 and T1. According to the above method, the transmittance change at 15 s and 2 min 15 s after the standard and the applied bacterial solution were mixed was detected, recorded as T2 and T3. Each sample was tested three times. The lysozyme content of the sample was calculated according to the following formula: lysozyme content (μg / mgprot) = (sample △T ÷ standard △T) × standard concentration ÷ sample protein concentration (mgprot / mL).

[0061] Each fusion protein pCZN1-WpDestabilase, pCZN1-WpdestabilaseC10 and

[0062] The activity detection results of pET32a-Trx-EK-WpDestabilase are shown in the following table.

[0063]

[0064] As shown in the table above, the antibacterial activity and thrombolytic activity of pCZN1-WpDestabilase fusion protein were significantly improved compared with pET32a-Trx-EK-wpDestabilase fusion protein. The isopeptidase activity of pCZN1-WpDestabilaseC10 fusion protein was significantly improved compared with pCZN1-WpDestabilase fusion protein, and the ability to dissolve old thrombi was significantly increased.

[0065] The preferred embodiments of the present invention are described in detail above. The detailed description of the technical scheme of the present invention with the help of the optimized embodiment is illustrative rather than restrictive. It cannot be determined that the specific embodiments of the present invention are limited to this. For ordinary technicians in the technical field to which the present invention belongs, without departing from the concept of the present invention, modifications to the technical schemes recorded in the embodiments or equivalent replacement of some of the technical features therein should be deemed to fall within the scope of patent protection determined by the claims submitted by the present invention.

Claims

1. A destabilizing enzyme fusion protein with enhanced activity, the amino acid sequence of the destabilizing enzyme fusion protein is shown in SEQ ID No.

5.

2. A biological material comprising the destabilizing enzyme fusion protein or a gene encoding the destabilizing enzyme fusion protein according to claim 1.

3. The biomaterial according to claim 2, characterized in that The biological material is selected from: A vector comprising the destabilizing enzyme fusion protein or a gene encoding it according to claim 1, or a host cell comprising the destabilizing enzyme fusion protein or a gene encoding it according to claim 1.

4. The biomaterial according to claim 3, characterized in that The vectors include cloning vectors and expression vectors.

5. The biomaterial according to claim 4, characterized in that The vector is pCZN1.

6. The biomaterial according to claim 3, characterized in that The host cell is a prokaryotic cell or a eukaryotic cell.

7. Use of the destabilizing enzyme fusion protein according to claim 1 or the biomaterial according to any one of claims 2 to 6 in thrombus dissolution and / or bacteriostasis.

8. Use of the destabilizing enzyme fusion protein according to claim 1 or the biomaterial according to any one of claims 2 to 6 in the preparation of an agent for dissolving thrombus and / or inhibiting bacteria.

9. A method for preparing the destabilizing enzyme fusion protein according to claim 1, characterized in that: The method comprises the step of expressing the destabilizing enzyme fusion protein according to claim 1 by using an expression vector.

10. The method according to claim 9, characterized in that The method further comprises the step of isolating / purifying the destabilized enzyme fusion protein.

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