Modifying enzyme, nucleic acid molecule, expression vector and application thereof

Abstract

The invention relates to the field of bioengineering, in particular to a modifying enzyme, a nucleic acid molecule, an expression vector and application thereof. The invention provides a modifying enzyme which has an amino acid sequence as shown in SEQ ID NO: 1. The conotoxin is modified by a biological modification enzyme, the problems in a chemical synthesis method are solved, and the biological activity and stability of the modified conotoxin are improved.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a modification enzyme, a nucleic acid molecule, an expression vector and applications thereof. Background Art

[0002] Conotoxins are a class of potent neurotoxins produced by cone snails (a marine gastropod mollusk). These toxins are mainly used to hunt small fish and other marine life. According to their structure and mechanism of action, conotoxins can be divided into several families, such as α-conotoxin, ω-conotoxin, μ-conotoxin, etc. Each family targets different nerve receptors or ion channels and has a specific mode of action: 1. α-conotoxin: mainly acts on nicotinic acetylcholine receptors to prevent the transmission of nerve signals. 2. ω-conotoxin: specifically blocks voltage-gated calcium channels and affects the release of neurotransmitters. 3. μ-conotoxin: acts on sodium channels, causing muscle paralysis.

[0003] Conotoxins have become a frontier in the development of new drugs due to their high affinity and selectivity for biological targets. However, current research focuses more on the discovery of toxin sequences, and less on the maturation, processing and modification mechanisms of toxins, which has restricted the progress of their drug applications.

[0004] Existing chemical modification methods are complex, costly, and have a significant impact on the environment. Therefore, it is of great significance to develop bioenzyme modification methods that can replace chemical modification. Summary of the invention

[0005] In view of this, the present invention provides a modification enzyme, a nucleic acid molecule, an expression vector and its application. The present invention modifies conotoxin by a biological modification enzyme, solves the problems in the chemical synthesis method, and improves the biological activity and stability of the modified conotoxin.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a modified enzyme having:

[0008] (1) the amino acid sequence shown in SEQ ID NO: 1; or

[0009] (2) An amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or

[0010] (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

[0011] In some embodiments of the present invention, the sequence shown in SEQ ID NO: 1 is: MAVPGFRPRCFFDVSIGDQPGGRIIFELFSEECPKTCENFRALCTGEKGVSEKTGETLHYKGAPFHRVVKDFMVQSGDFTKGDGTGGESIYGGHFPDENFKFKHDKEFLLSMANRGKDTNGSQFFM.

[0012] The present invention also provides a nucleic acid molecule encoding the above-mentioned modification enzyme, which has:

[0013] (4) the nucleotide sequence shown in SEQ ID NO: 2; or

[0014] (5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as the nucleotide sequence shown in (4); or

[0015] (6) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (4) or (5).

[0016] In some embodiments of the invention, as SEQ ID The sequence shown in NO:2 is: ATGGCGGTTCCGGGCTTCCGTCCGCGTTGTTTCTTCGATGTTTCTATCGGCGATCAGCCGGGCGGCCGTATCATCTTCGAACTGTTCAGCGAAGAATGCCCGAAAACCTGCGAAAACTTCCGTGCGCTGTGCACCGGCGAAAAAGGCGTTAGCGAAAAAACCGGTGAAACCCTGCACTACAAAGG CGCGCCGTTCCACCGTGTTGTTAAAGATTTCATGGTTCAGTCTGGCGACTTTACTAAAGGGTGACGGTACCGGTGGTGAGTCCATCTACGGCGGCCACTTCCCGGATGAAAACTTTAAGTTTAAACATGATAAAGAATTCCTACTGAGCATGGCGAACCGTGGTAAAGATACCAACGGTAGCCAGTTCTTCATGTAA.

[0017] The present invention also provides an expression cassette, comprising: the above nucleic acid molecule.

[0018] The present invention also provides an expression vector, comprising: the above nucleic acid molecule and / or the above expression cassette.

[0019] The present invention also provides a host for transforming and / or transfecting the above expression vector.

[0020] In some embodiments of the present invention, the above-mentioned host, transformed and / or transfected chassis strain includes: Yarrowia lipolytica.

[0021] The present invention also provides the use of the above-mentioned modification enzyme, the above-mentioned nucleic acid molecule, the above-mentioned expression cassette, the above-mentioned expression vector and / or the above-mentioned host in regulating the toxicity of conopeptide.

[0022] The present invention also provides the use of the above-mentioned modification enzyme, the above-mentioned nucleic acid molecule, the above-mentioned expression cassette, the above-mentioned expression vector and / or the above-mentioned host in the preparation of products containing conopeptide.

[0023] The present invention also provides a product, comprising: the above-mentioned modification enzyme, the above-mentioned nucleic acid molecule, the above-mentioned expression cassette, the above-mentioned expression vector and / or the above-mentioned host.

[0024] In some embodiments of the present invention, the above-mentioned product includes: a drug.

[0025] The invention provides a biological modification enzyme, which is heterologously expressed in Yarrowia lipolytica by genetic engineering technology, and its modification effect on cono polypeptide is proved by cell biology experiments. The enzyme can be combined with cono polypeptide, promote its post-translational modification, and then change its biological activity. The modified conotoxin can effectively reduce the vitality of HT22 cells, induce ROS generation and oxidative stress reaction, and cause cell apoptosis. In addition, the modified toxin also shows the effect of calcium ion accumulation, which further enhances its cytotoxicity. Through this biological modification method, not only the stability and specificity of the toxin are improved, but also the production cost and environmental pollution are greatly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0027] Figure 1 The figure shows the effect of different concentrations of conus peptides on cell survival rate; among them: polf-4C is the control group, which is the blank control of engineering bacteria; 248E is the modified peptide; S-48 is the peptide without adding modification enzyme;

[0028] Figure 2 The effect of cono peptides on cellular redox levels is shown;

[0029] Figure 3 The effect of modified cono polypeptide on cell morphology and structure is shown; wherein: A shows cells of the control group; B shows cells of the modified polypeptide treatment group;

[0030] Figure 4The effect of modified cono polypeptide on cell apoptosis is shown; from left to right are the control group, S-48 is a polypeptide without adding modifying enzyme, and 248E is a modified polypeptide. DETAILED DESCRIPTION

[0031] The invention discloses a modification enzyme, a nucleic acid molecule, an expression vector and applications thereof.

[0032] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0033] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0034] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0035] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0036] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0037] The modification effect of the conotoxin modifying enzyme provided by the present invention on cono polypeptides is evaluated by cell biology experiments to evaluate the biological activity of the modified conotoxin on HT22 cells, including cytotoxicity, oxidative stress response, ROS generation, Ca²⁺ accumulation and apoptosis induction.

[0038] Through the present invention, we have verified that the discovered conotoxin modifying enzyme can effectively modify cono polypeptide to produce cytotoxicity. The modified toxin can induce the death of HT22 cells through multiple pathways (ROS generation, apoptosis, change of cell structure, etc.), indicating that the modified enzyme has great application potential in drug development, especially in the development of anticancer drugs and neuromodulatory drugs. The next step of the study will be to further optimize the reaction conditions of the enzyme and explore its application in other biological models.

[0039] In Example 1 and Effect Example 1 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0040] The present invention will be further described below in conjunction with embodiments:

[0041] Example 1

[0042] 1. Expression and purification of modification enzymes

[0043] The expression vector of conotoxin-modifying enzyme was constructed by using genetic engineering technology, and it was co-expressed with cono polypeptide in Yarrowia lipolytica. The expressed cono polypeptide was purified by ultrafiltration, and the purified enzyme was identified and analyzed by SDS-PAGE electrophoresis.

[0044] The gene sequence was optimized and synthesized by Sangon Biotech Co., Ltd. according to the codons of Yarrowia lipolytica, amplified from the vector with the synthetic gene, obtained by PCR using KOD OneTM PCR Master Mix enzyme (TOYOBO, JAPAN), and connected to the yeast expression plasmid backbone by homologous recombination using ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co., Ltd, Nanjing). The plasmid was obtained. Positive clones were obtained, the strains were expanded, and the cells were collected and lysed, and the conotoxin polypeptide was extracted using an ultrafiltration tube.

[0045] 2. Cytotoxicity Assay

[0046] The effect of conotoxin on HT22 cell viability was detected by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylphenyl)-2H-tetrazolyl monosodium salt) (WST-8) (Biosharp, BS350A). Cells were seeded in 96-well plates with a total of 100 μL of culture medium (cells were seeded at a starting density of 20%) and cultured for 20 h (the confluence reached 70-80%). Immediately thereafter, 100 μL of conotoxin culture medium prepared in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin was added to obtain treatment concentrations (0, 25, 50, 100 μM), respectively. After incubation for 24 h, 10 μL of CCK-8 solution was added to each well and incubated at 37 °C for 1 h. The absorbance was measured at 450 nm using a spectrophotometer.

[0047] 3. ROS generation detection

[0048] According to the cell viability assay, the subsequent experiments used a treatment concentration of 50 μM. HT22 cells were seeded in 24-well plates (seeded at a starting density of 20%), cultured for 24 h, treated with 50 μM conotoxin for 12 h, and collected by 0.25% (w / v) trypsin digestion and washed twice with PBS. ROS was determined by 2′, 7′-dichlorodihydrofluorescein (DCFH-DA) (Beyotime, S0033S) staining. That is, cells were incubated with 10 μmol L -1 The PBS solution of DCFH-DA was incubated at 37°C in the dark for 20 min, then washed twice with PBS. The intensity of dichlorofluorescein (DCF) was used to estimate the generation of ROS using flow cytometry (BECKMAN, USA).

[0049] 4. Cell apoptosis detection

[0050] FITC (Biosharp, BL110A) staining was used to detect cell apoptosis. HT22 cells were seeded in 6-well plates at a starting density of 30%, cultured for 24 h, treated with 50 μM conotoxin for 12 h, and then digested with trypsin to collect the cells and washed twice with PBS. 5 Each cell was resuspended in binding buffer, stained with 5 μL Annexin-V FITC and 10 μL propidium iodide (PI), and then incubated for 15 min at room temperature in the dark according to the instructions. The cell apoptosis rate was detected by flow cytometry (BECKMAN, USA).

[0051] 5. Cell morphology observation

[0052] To observe the changes in cell morphology, a cell imaging system was used for observation. HT22 cells were seeded in a 24-well plate at a starting density of 20%, cultured for 24 h, treated with 50 μM conotoxin for 12 h, and cell images were obtained using a microscope (10× / 20×).

[0053] Effect Example 1 Experimental Results

[0054] 1. Obtaining the protein sequence of the modification enzyme and constructing the expression system:

[0055] Based on the omics data of Conus snails, we screened and obtained a sequence of peptidylprolyl isomerase G (PPIG) for study. After codon optimization, we constructed an expression vector for Yarrowia lipolytica.

[0056] 2. Cell viability and toxicity assay

[0057] The results of MTT assay showed that the modified conotoxin significantly reduced the survival rate of HT22 cells in a dose-dependent manner (p<0.01). Compared with the unmodified peptide (S-48), the cytotoxicity of the modified conotoxin was enhanced by about 2 times.

[0058] Table 1 Cell survival rate

[0059]

[0060] 3. ROS generation detection

[0061] The results of ROS level detection showed that the ROS level of HT22 cells in the modified conotoxin treatment group was significantly increased, indicating that it induced oxidative stress response.

[0062] Table 2 Cell survival rate

[0063]

[0064] 4. Cell morphology analysis

[0065] Cell imaging results showed that the control group (such as Figure 3 A) Living cells usually have a transparent appearance and intact cell membrane, while the modified conotoxin-treated groups (such as Figure 3 (B) The cell morphology changed, indicating that conotoxin had a significant inhibitory effect on the cells.

[0066] 5. Cell apoptosis detection

[0067] Flow cytometry results showed that the apoptosis rate of cells in the modified conotoxin-treated group increased significantly in a dose-dependent manner. Compared with the unmodified control group, the modified conotoxin induced a higher apoptosis rate.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A modified enzyme, characterized in that It has: (1) the amino acid sequence shown in SEQ ID NO: 1; or (2) An amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

2. A nucleic acid molecule encoding the modification enzyme according to claim 1, characterized in that: It has: (4) the nucleotide sequence shown in SEQ ID NO: 2; or (5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as the nucleotide sequence shown in (4); or (6) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (4) or (5).

3. An expression cassette, characterized in that include: The nucleic acid molecule according to claim 2.

4. An expression vector, characterized in that include: The nucleic acid molecule according to claim 2 and / or the expression cassette according to claim 3.

5. A host, characterized in that Transformation and / or transfection with the expression vector according to claim 4.

6. The host according to claim 5, characterized in that Chassis strains for transformation and / or transfection include: Yarrowia lipolytica.

7. Use of the modification enzyme according to claim 1, the nucleic acid molecule according to claim 2, the expression cassette according to claim 3, the expression vector according to claim 4 and / or the host according to claim 5 or 6 in regulating the toxicity of conopeptides.

8. Use of the modification enzyme according to claim 1, the nucleic acid molecule according to claim 2, the expression cassette according to claim 3, the expression vector according to claim 4 and / or the host according to claim 5 or 6 in the preparation of a product containing conopeptide.

9. A product, characterized in that include: The modification enzyme according to claim 1, the nucleic acid molecule according to claim 2, the expression cassette according to claim 3, the expression vector according to claim 4 and / or the host according to claim 5 or 6.

10. The product according to claim 9, characterized in that include: drug.

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