A wide-body gold leech unstable enzyme fusion protein and application thereof

Abstract

The application discloses a destabilized enzyme fusion protein with improved activity, and the amino acid sequence of the destabilized enzyme fusion protein is shown in SEQ ID No. 3 or SEQ ID No. 5. The application improves the bacteriostatic activity, thrombolytic activity and isopeptidase activity of the destabilized enzyme fusion protein by optimizing and reengineering the expression strategy of the destabilized enzyme Wpdestabilase from Whitman's broad body leech, and improves the application prospect and application space of the Wpdestabilase.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an unstable enzyme fusion protein derived from the broad-bodied golden leech and its applications. Background Technology

[0002] The broad-bodied golden leech (Whitmania pigra Whitman) belongs to the order Hirudinales, family Hirudinidae, and genus Whitmania. It is one of the basal animals of the Chinese medicinal leech and has significant medicinal value. It is widely distributed in my country and inhabits paddy fields and ditches of the same type year-round, feeding on the body fluids of fresh snails.

[0003] The applicant screened a transcript of a leech polypeptide with antibacterial and hemolytic effects from the *Hirudo pigra* salivary gland transcriptome database (SRX16840326, SRX16840327, SRX16840328), and verified the sequence correctness by cDNA sequence cloning. After removing the polypeptide signal peptide, full-length splicing primers were designed based on the PAS method, the target gene was synthesized, and 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, disruption, and purification, the fusion target protein with N-terminal Trx tags was obtained by SDS-PAGE. The antibacterial activity of the fusion target protein was 14.17 μg / mL, and the hemolysis rate was 44.06%. For details, please refer to Chinese Invention Patent, Application No.: CN202111590989.2; Authorization Announcement No.: CN114057866B.

[0004] To further enhance the activity of the broad-bodied golden thread leech destabilase (WpDestabilase), the applicant optimized the expression strategy of WpDestabilase, obtaining a fusion protein with significantly enhanced antibacterial and thrombolytic activities. Summary of the Invention

[0005] On the one hand, the present invention provides an unstable enzyme fusion protein with enhanced activity, the amino acid sequence of which is shown in SEQ ID No. 3 or SEQ ID No. 5.

[0006] In one embodiment, the amino acid sequence of the unstable enzyme fusion protein is shown in SEQ ID No. 3.

[0007] In one embodiment, the amino acid sequence of the unstable enzyme fusion protein is shown in SEQ ID No. 5.

[0008] On the other hand, the present invention also provides the encoding gene of the above-mentioned unstable enzyme fusion protein.

[0009] In one embodiment, the sequence of the gene encoding the unstable enzyme fusion protein is shown in SEQ ID No. 4.

[0010] In one embodiment, the sequence of the gene encoding the unstable enzyme fusion protein is shown in SEQ ID No. 6.

[0011] On the other hand, the present invention also provides biological materials comprising the above-mentioned unstable enzyme fusion protein or its encoding gene. The biological material is selected from: vectors comprising the above-mentioned unstable enzyme fusion protein or its encoding gene, or host cells comprising the above-mentioned unstable enzyme fusion protein or its encoding gene.

[0012] On the other hand, the present invention also provides a vector containing the above-mentioned encoding gene, or a host cell containing the vector.

[0013] In one embodiment, the vector includes a cloning vector and an expression vector.

[0014] In one embodiment, the carrier is pCZN1.

[0015] In one embodiment, the carrier is ring-shaped or linear.

[0016] As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different host cells. "Expression vector" refers to a vector capable of incorporating and expressing heterologous DNA fragments into foreign cells. Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of a suitable expression vector is well known to those skilled in the art.

[0017] In one embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0018] In one embodiment, the prokaryotic cells are derived from Escherichia coli or Agrobacterium.

[0019] In one embodiment, the eukaryotic cell is yeast.

[0020] On the other hand, the present invention also provides the use of the above-mentioned unstable enzyme fusion protein, or biomaterials containing the above-mentioned unstable enzyme fusion protein or its encoding gene, in thrombolysis and / or antibacterial activity.

[0021] On the other hand, the present invention also provides the use of the above-mentioned unstable enzyme fusion protein, or biological material containing the above-mentioned unstable enzyme fusion protein or its encoding gene, in the preparation of reagents for dissolving thrombi and / or inhibiting bacteria.

[0022] In one embodiment, the antibacterial effect is the inhibition of cocci, such as Micrococcus luteus.

[0023] In one embodiment, the thrombus is selected from one or more of fresh thrombi and aged thrombi (or, old thrombi).

[0024] On the other hand, the present invention also provides a method for preparing the above-mentioned unstable enzyme fusion protein, the method comprising the step of expressing the above-mentioned unstable enzyme fusion protein using an expression vector.

[0025] Furthermore, the method also includes the step of isolating / purifying the unstable enzyme fusion protein.

[0026] This invention improves the antibacterial, thrombolytic, and isopeptidase activities of the unstable enzyme WpDestabilase fusion protein by optimizing the expression strategy of WpDestabilase derived from broad-bodied golden leech, thereby enhancing the application prospects and scope of WpDestabilase. Attached Figure Description

[0027] Figure 1 Figure 1 shows the expression, purification, and refolding results of the pCZN1-WpDestabilase fusion protein. a) Expression of the pCZN1-WpDestabilase fusion protein: M: protein marker; Lane 1: CK (complete cell lysate after pCZN1 empty vector induction); Lane 2: CK (without IPTG); Lane 3: after induction; Lane 4: supernatant after induction and lysis; Lane 5: precipitate after induction and lysis. b) Purification of the pCZN1-WpDestabilase fusion protein: M: protein marker; Lane 1: precipitate of the pCZN1-WpDestabilase fusion protein after induction and lysis; Lane 2: flowing liquid; Lanes 3 and 4: proteins obtained by elution. c) Protein analysis after refolding of the pCZN1-WpDestabilase fusion protein: M: protein marker; Lane 1: detection results of the refolded and purified pCZN1-WpDestabilase fusion protein.

[0028] Figure 2Figure 1 shows the expression, purification, and refolding results of the pCZN1-WpDestabilaseC10 fusion protein. a) Expression of the pCZN1-WpDestabilaseC10 fusion protein: M: protein marker; Lane 1: CK (complete cell lysate after pCZN1 empty vector induction); Lane 2: CK (without IPTG); Lane 3: after induction; Lane 4: supernatant after induction and lysis; Lane 5: precipitate after induction and lysis. b) Purification of the pCZN1-WpDestabilaseC10 fusion protein: M: protein marker; Lane 1: precipitate after pCZN1-WpDestabilaseC10 induction and lysis; Lane 2: flowing liquid; Lanes 3 and 4: proteins obtained by elution. c) Protein analysis after refolding of the pCZN1-WpDestabilaseC10 fusion protein. M: Protein marker; Lane 1: Detection results of refolded and purified pCZN1-WpDestabilaseC10 fusion protein. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make modifications to the disclosed technical content to create equivalent embodiments. Unless otherwise specified, the materials, reagents, instruments, and methods used in the following embodiments are all conventional materials, reagents, instruments, and methods in the art and are commercially available.

[0030] Any simple modifications or equivalent changes made to the following embodiments without departing from the scope of the present invention and based on the technical essence of the present invention shall fall within the protection scope of the present invention.

[0031] Example 1: Expression, purification, and refolding of pCZN1-WpDestabilase fusion protein

[0032] As described in the aforementioned patent (Chinese invention patent application, application number: CN202111590989.2), the amino acid sequence of the destabilizing enzyme WpDestabilase from the broad-bodied golden leech is as follows (SEQ ID No. 1):

[0033] MMKSAIYSCFALLTIVLALSEVNS QISDPCLRCICKEEGCETQIGQCNDGTSQSCGPYQ IMRAYWIDCGKPGNDYETCTKTIDCSEACVRAYMNRYGTYCTGGRTPTCQDYARIHKGGPSGCNQSETFVYGKKVQECSVIPATETTTEI (underlined part is signal peptide).

[0034] The amino acid sequence of the pET32a-Trx-EK-WpDestabilase fusion target protein used in the above patent is shown below (SEQ ID No. 2):

[0035] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQ HMDSPDLGTDDDDKAMASQISDPCLRCICKEEGCETQIGQCNDGTSQSCGPYQIMRAYWIDCGKPGNDYETCTKTIDCSEACVRAYMNRYGTYCTGGRTPTCQDYARIHKGGPSGCNQSETFVYGKKVQECSVIPATETTTEI.

[0036] The pET32a-Trx-EK-wpDestabilase protein has its N-terminus fused with the TrxA, His, thrombin recognition site, S-tag, and enterokinase (EK) recognition site sequences of the pET32a vector.

[0037] In this embodiment, in order to improve the activity of the WpDestabilase fusion protein, an attempt was made to reduce the amino acid sequence of non-target peptides by changing the expression strategy.

[0038] In this embodiment, the pCZN1 expression vector was used to express WpDestabilase, reducing the number of redundant amino acid sequences beyond the target polypeptide. The amino acid sequence of the resulting fusion protein pCZN1-WpDestabilase is shown below (SEQ ID No. 3), and its gene sequence is shown in SEQ ID No. 4.

[0039] MNHKVHHHHHHMQISDPCLRCICKEEGCETQIGQCNDGTSQSCGPYQIMRAYWIDC GKPGNDYETCTKTIDCSEACVRAYMNRYGTYCTGGRTPTCQDYARIHKGGPSGCNQSET FVYGKKVQECSVIPATETTTEI (SEQ ID No. 3).

[0040] ATGAATCACAAAGTGCATCATCATCATCATCATATGCAGATCAGTGATCCGTGTCTGCGCTGTATTTGTAAAGAAGAAGGTTGTGAAACCCAGATTGGTCAGTGCAATGATGGCACCAGTCAGAGTTGTGGTCCGTATCAGATTATGCGTGCATATTGGATTGACTGTGGCAAACCGGGTAATGATTATGAAACCTGCACCAAAACCAT CGATTGTAGTGAAGCATGTGTGCGTGCATATATGAATCGTTATGGCACCTATTGTACCGGCGGTCGTACCCCTACCTGTCAGGATTATGCCCGTATTCATAAAGGTGGTCCGAGTGGTTGTAATCAGAGCGAAACCTTTGTTTATGGCAAAAAAGTGCAGGAATGCAGCGTGATTCCGGCAACCGAAACCACCACCGAAATTTAA(SEQ ID No.4).

[0041] To further modify the amino acid sequence of the target polypeptide, the 10 amino acid sequence “IPATETTTEI” at the C-terminus of the WpDestabilase target polypeptide was deleted in this embodiment. This sequence is denoted as pCZN1-WpDestabilaseC10, and its amino acid sequence is shown below (SEQ ID No. 5). Its gene sequence is shown in SEQ ID No. 6.

[0042] MNHKVHHHHHHMQISDPCLRCICKEEGCETQIGQCNDGTSQSCGPYQIMRAYWIDC GKPGNDYETCTKTIDCSEACVRAYMNRYGTYCTGGRTPTCQDYARIHKGGPSGCNQSET FVYGKKVQECSV (SEQ ID No. 5).

[0043] ATGAATCACAAAGTGCATCATCATCATCATCATATGCAGATCAGCGATCCGTGTCTGCGTTGCATTTGCAAAGAAGAAGGCTGCGAAACCCAGATTGGTCAGTGCAATGATGGTACCAGTCAGAGTTGCGGTCCGTATCAGATTATGCGCGCATATTGGATTGATTGTGGCAAACCGGGTAATGATTATGAAACCTGTACCAAAACCATCGATTGCAGCGAAGCCTGTGTGCGTGCATATATGAATCGCTATGGTACCTATTGCACCGGTGGTCGTACCCCGACCTGTCAAGATTATGCCCGCATTCATAAAGGCGGTCCGAGTGGTTGTAATCAGAGTGAAACCTTTGTGTATGGCAAAAAAGTGCAGGAATGCAGCGTTTAA(SEQ ID No.6)。

[0044] Specifically, in this embodiment, the target polypeptide signal peptide sequence was removed from the destabilizing enzyme WpDestabilase derived from *Hirudo medicinalis*, and the target gene sequence was artificially synthesized and ligated into the pCZN1 plasmid to construct the pCZN1-WpDestabilase recombinant plasmid. The validated recombinant plasmid was introduced into competent *E. coli* (Arctic-Express) bacteria to construct a WpDestabilase prokaryotic expression system. *E. coli* bacteria containing the WpDestabilase recombinant plasmid were activated on LB agar plates. Selected single colonies were inoculated into test tubes containing 3 mL of LB medium with 50 μg / mL Amp and incubated overnight at 37°C with shaking at 200 rpm. The next day, the inoculum was diluted 1:100 in 100 mL of LB medium with 50 μg / mL Amp and incubated at 37°C with shaking at 200 rpm until the bacterial OD600 reached 0.6-0.8. Take 1 mL of culture, centrifuge at 10000 rpm for 2 min at room temperature, 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 rpm to induce fusion protein expression. Take 1 mL of culture, centrifuge at 10000 rpm for 2 min at room temperature, discard the supernatant, and resuspend the bacterial pellet in 100 μL of 1× loading buffer. Centrifuge the remaining culture at 4000 rpm for 10 min, discard the supernatant, and resuspend the bacterial pellet in PBS; after sonication of the resuspended solution, resuspend the supernatant and pellet separately in loading buffer. Perform 12% SDS-PAGE analysis, and stain with Coomassie Brilliant Blue for banding.

[0045] 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 rpm 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 rpm for 15 min at room temperature. The supernatant was collected.

[0046] The steps for Ni-column affinity purification of the refolded fusion protein are as follows: 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 OD280 value of the eluent reached baseline. The column was then washed 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 OD280 value of the eluent reached baseline. Finally, 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 then performed.

[0047] The expression, purification, and refolding results of the pCZN1-WpDestabilase fusion protein are as follows: Figure 1 As shown in the figure. The expression, purification, and refolding results of the pCZN1-WpdestabilaseC10 fusion protein are as follows. Figure 2 As shown.

[0048] Example 2: Verification of the activity of the WpDestabilase fusion protein

[0049] In this embodiment, the thrombolytic activity, isopeptidase activity, fibrin platelet dissolving activity, and lysozyme activity of the pCZN1-WpDestabilase and pCZN1-WpDestabilaseC10 fusion proteins obtained in Example 1 were detected. Simultaneously, aspirin, PBS, and the enzyme described in Chinese invention patent application CN202111590989.2 were also tested.

[0050] The activity of the pET32a-Trx-EK-WpDestabilase fusion protein.

[0051] 2.1 Detection of in vitro thrombolytic activity

[0052] 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°C for 1 hour, the fresh blood clot was removed. The clot was rinsed with physiological saline until the liquid was colorless, then placed in a 37°C incubator for 1 hour. The clot was then removed, turned over, and placed back in the 37°C incubator for another 1 hour to obtain a fresh thrombus. Fresh thrombi stored at 4°C for 48 hours are considered old thrombi (Wyshelesky et al., 2001).

[0053] Add 0.1g of fresh or old thrombus to 1mL of test sample and place in a 37℃ constant temperature shaker at 60r / min for 48h. Remove the remaining thrombus, rinse the surface of the blood clot with physiological saline to remove impurities, and place in an oven at 37℃ for 1h. After turning the blood clot over, treat it again at 37℃ for 1h, weigh it, and calculate the thrombolysis rate according to the following formula. Each group was repeated 3 times. Thrombolysis rate = [(mass of thrombus before dissolution - mass of thrombus after dissolution) / mass of thrombus before dissolution] × 100%.

[0054] 2.2 Detection of isopeptidase activity

[0055] 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 is measured, and the difference in absorbance before and after the reaction is taken as the isopeptidase activity of the 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 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, and PBS without the sample was used as a control. The assay was repeated three times.

[0056] 2.3 Detection of fibrin plate dissolution effect

[0057] Mix 20 mL of 1% agarose solution and 8 mL of 0.5% fibrinogen solution, then add 1 mL of 10 U / mL thrombin solution. Stir well and immediately pour into a sterile petri dish (9 cm in diameter). Gently shake to mix, and allow to cool for about 30 minutes until the agar is completely solidified to prepare fibrin plates. Punch wells (8 mm in diameter) on the fibrin plates, with a well spacing of more than 1.5 cm. Add 5 μL of test sample to each well, incubate overnight at 37°C, and observe the dissolution zone. Repeat 5 times.

[0058] 2.4 Detection of lysozyme activity

[0059] 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 following formula: Lysozyme content (μg / mgprot) = (Sample ΔT ÷ Standard ΔT) × Standard concentration ÷ Sample protein concentration (mgprot / mL).

[0060] Each fusion protein pCZN1-WpDestabilase, pCZN1-WpdestabilaseC10 and

[0061] The activity assay results of pET32a-Trx-EK-WpDestabilase are shown in the table below.

[0062]

[0063] As shown in the table above, the pCZN1-WpDestabilase fusion protein exhibits significantly enhanced antibacterial and thrombolytic activities compared to the pET32a-Trx-EK-wpDestabilase fusion protein. The pCZN1-WpDestabilaseC10 fusion protein, compared to the pCZN1-WpDestabilase fusion protein, shows significantly increased isopeptidase activity and a significantly enhanced ability to dissolve old thrombi.

[0064] The preferred embodiments of the present invention have been described in detail above. The detailed description of the technical solutions of the present invention by means of optimized embodiments is illustrative and not restrictive. It should not be considered that the specific embodiments of the present invention are limited to these. For those skilled in the art, any modifications to the technical solutions described in the embodiments or equivalent substitutions of some technical features without departing from the concept of the present invention should be considered as falling within the scope of patent protection defined by the claims of the present invention.

Claims

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

3.

2. Biological material comprising the gene encoding the unstable enzyme fusion protein of claim 1.

3. The biomaterial according to claim 2, characterized in that, The biomaterials are selected from: A vector containing the encoding gene of the unstable enzyme fusion protein of claim 1, or a host cell containing the encoding gene of the unstable enzyme fusion protein of claim 1.

4. The biomaterial according to claim 3, characterized in that, The carrier is an expression carrier.

5. The biomaterial according to claim 4, characterized in that, The carrier 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 unstable enzyme fusion protein of claim 1 or the biomaterial of any one of claims 2-6 in the preparation of a reagent for dissolving thrombi.

8. A method for preparing the unstable enzyme fusion protein according to claim 1, characterized in that, The method includes the step of expressing the encoding gene of the unstable enzyme fusion protein of claim 1 using an expression vector.

9. The method according to claim 8, characterized in that, The method further includes the step of isolating / purifying the unstable enzyme fusion protein.

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