Superoxide dismutase HSTM-SOD and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to superoxide dismutase HSTM-SOD and application thereof. The amino acid sequence of the superoxide dismutase HSTM-SOD provided by the invention is selected from at least one of sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 8, or a sequence with more than 80% of homology with the amino acid sequence. The HSTM-SOD with different sequences, which is provided by the invention, generally has excellent thermal stability, pH stability, good compatibility and super-strong membrane crossing capability; the HSTM-SOD shows remarkable capabilities in the aspects of promoting cell sunburn repair, promoting cell oxidative damage repair, resisting ferroptosis caused by TalaA, promoting wound healing of diabetic patients, improving age-related macular degeneration and the like, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to superoxide dismutase HSTM-SOD and application thereof. BACKGROUND

[0002] Superoxide dismutase (SOD) is a kind of vital antioxidant enzyme existing in organisms, and is also a special scavenger of superoxide anion free radicals (·O2 - ) recognized internationally, which can effectively prevent the damage of superoxide free radicals to the body, and plays a vital role in balancing the function of the body's oxidation and antioxidant system and relieving free radical damage.

[0003] At present, the production of SOD is mainly extracted from animal blood or liver, but it is difficult to popularize and apply due to limited raw material sources and easy pollution. Although SOD has experienced technical updates from 'animal extraction, plant extraction' to'microbial extraction, chemical modification', the most critical 'neck-breaking technology' has not been substantially broken through, and the development of SOD and its related downstream industry has been severely restricted.

[0004] Most importantly, SOD is a kind of biological enzyme, and has the problem of instability after being separated from the body, which leads to the fact that many terminal products added with SOD have SOD activity at the beginning, but the activity is very little when finally reaching the hands of consumers after storage and transportation, which greatly limits the application of SOD. SUMMARY

[0005] In order to solve the above technical problems, the application provides a superoxide dismutase HSTM-SOD and application thereof.

[0006] In the application, the inventors select an amino acid sequence with strong protein skeleton rigidity from a protein library by using a computer, find out the best primary structure in a way of replacing with simulated amino acid, design a coding codon in a way of reverse transcription, perform whole gene synthesis, finally insert into a pET system expression vector for expression, and obtain a series of superoxide dismutases HSTM-SOD with good enzymatic properties.

[0007] The superoxide dismutase HSTM-SOD provided by the application has an amino acid sequence selected from at least one of sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or a sequence with more than 80% homology with the above amino acid sequence.

[0008] As a preferred, the solution of the dissolved superoxide dismutase HSTM-SOD is red to red-brown in appearance, and the color depth is positively correlated with the concentration of the solution.

[0009] Meanwhile, the gene encoding the superoxide dismutase HSTM-SOD is also the focus of the present application.

[0010] In addition, the recombinant expression vector containing the superoxide dismutase HSTM-SOD and the recombinant cell obtained by transforming the expression vector into the recipient cell also fall within the scope of the present application.

[0011] Further, the present application also provides the application of the superoxide dismutase HSTM-SOD or the recombinant expression vector, and more specifically, the application includes but is not limited to the following aspects: (1) the application in the preparation of products for resisting cell iron death related diseases; (2) the application in the preparation of products resistant to digestive enzyme enzymolysis; (3) the application in the preparation of products for promoting wound healing in diabetic patients; (4) the application in the preparation of cell damage repair products; (5) the application in the preparation of products for relieving ocular fundus cell oxidative damage.

[0012] In the above application, as a preferred, the product in (1) is any one of the products for preventing hair loss caused by hair follicle cell iron death or hair follicle necrosis.

[0013] As a preferred, the cell damage in (4) includes but is not limited to ultraviolet (UV) induced cell damage.

[0014] The experimental results of the present application show that the superoxide dismutase HSTM-SOD of different sequences provided by the present application has good repair effect on UV induced human immortalized keratinocyte (HaCaT) cell damage, and HSTM-SOD can greatly rescue cell death caused by cell iron death inducer (TalaA); in addition, it also shows significant effect on cell regeneration and healing of diabetic wound, in short, HSTM-SOD shows good potential in antioxidant and cell damage protection and other aspects.

[0015] Further, the present application also provides a composition for cell protection and / or cell damage repair, which contains the superoxide dismutase HSTM-SOD or the recombinant expression vector.

[0016] Preferably, the composition includes superoxide dismutase HSTM-SOD and an amino acid, wherein the amino acid is at least one selected from N-acetyl-L-cysteine ​​and glutathione.

[0017] Preferably, the composition has at least one property such as promoting cell repair of sunburn, promoting cell repair of oxidative damage, and anti-oxidation. Furthermore, the composition includes, but is not limited to, cosmetics, health products, pharmaceuticals, food, and other categories, and is not limited to products in solid, liquid, or aerosol form.

[0018] The beneficial effects of this invention are as follows: Firstly, the superoxide dismutase HSTM-SOD provided by this invention has strong enzymatic properties, specifically as follows: Excellent thermal stability: Even at temperatures as high as 95°C, the superoxide dismutase HSTM-SOD provided by this invention can still exhibit more than 90% activity. Excellent pH tolerance: HSTM-SOD has a wide pH tolerance range and strong acid and alkali resistance. In the pH range of 5.0~10.0, the enzyme activity of HSTM-SOD still maintains 88% or even higher. Good surfactant compatibility: It maintains stable enzymatic activity in surfactants such as Triton and Tween. Even in a 2% concentration of sodium dodecyl sulfate, HSTM-SOD can still maintain its original activity, which is a capability that ordinary enzymes do not have.

[0019] Secondly, the superoxide dismutase HSTM-SOD provided by this invention has good cell protection and repair effects, specifically manifested in: The HSTM-SOD provided by this invention can induce upregulation of growth factor (EGFR, IGF1R, IGF2R) mRNA expression, thereby promoting cell viability and growth; it has a significant restorative effect on mouse cells damaged by ultraviolet irradiation; it has a wound-healing effect on diabetic patients; and HSTM-SOD shows a significant and unique ability to combat ferroptosis induced by TalaA, which is not possessed by other types of superoxide dismutases.

[0020] Thirdly, the superoxide dismutase HSTM-SOD provided by this invention has a strong membrane-crossing ability without the addition of a signal peptide, which is not possessed by ordinary superoxide dismutases. This is extremely beneficial to the downstream applications of superoxide dismutase and greatly expands its application scope. Attached Figure Description

[0021] Figure 1This is an enzyme activity diagram of HSTM-SOD with five amino acid sequences in Experimental Example 1 of this invention; Figure 2 This is a thermal stability diagram of the five amino acid sequences after HSTM-SOD high-temperature treatment in Experiment Example 2 of this invention; Figure 3 This is an enzyme activity diagram of HSTM-SOD after treatment at different temperatures in Experimental Example 3 of the present invention; Figure 4 This is a graph showing the membrane-crossing ability of HSTM-SOD in Experiment Example 4 of this invention; Figure 5 This is a graph showing the tolerance of HSTM-SOD to different pH values ​​in Experimental Example 5 of this invention. Figure 6 This is a graph showing the fermentation expression level of HSTM-SOD in Experiment Example 6 of this invention; Figure 7 This is the repair effect of HSTM-SOD on TalaA-induced cell death in Experimental Example 7 of the present invention; Figure 8 This is a diagram showing the repair effect of HSTM-SOD on UV-damaged cells in Experiment Example 8 of this invention; Figure 9 This is a cellular experimental diagram of HSTM-SOD's effect on the cell repair of UV sunburn in Experiment Example 8 of the present invention; Figure 10 This is a graph showing the RNA sequencing results of HSTM-SOD in the cell repair of UV-damaged cells in Experiment Example 8 of this invention. Figure 11 This is a graph showing the healing ability of HSTM-SOD on the wounds of diabetic patients in Experiment Example 9 of this invention. Figure 12 This is a diagram showing the rescue effect of HSTM-SOD on age-related macular degeneration in Experiment Example 10 of this invention. Detailed Implementation

[0022] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0023] Example 1 Superoxide dismutase HSTM-SOD was prepared using the following method: By screening protein libraries using a computer, amino acid sequences with strong protein backbone rigidity were selected. The optimal primary structure was found by using simulated amino acid substitution. Then, coding codons were designed using reverse transcription, and the whole gene was synthesized. After the gene ORF was synthesized, it was inserted into the pET series expression vectors, obtaining eight amino acid candidate sequences for superoxide dismutase HSTM-SOD. The eight amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

[0024] Experimental Example 1 We selected five superoxide dismutases (HSTM-SOD) with the above-mentioned amino acid sequences and investigated their enzyme activity.

[0025] The activity of superoxide dismutase based on the selected five amino acid sequences was determined according to the specifications in GB / T 5009.171-2003. Figure 1 As shown, Figure 1 In the above, HSTM-SOD-1 is SEQ ID NO:1, HSTM-SOD-2 is SEQ ID NO:2, HSTM-SOD-3 is SEQ ID NO:3, HSTM-SOD-4 is SEQ ID NO:4, and HSTM-SOD-5 is SEQ ID NO:5. The same applies to the following, and will not be repeated.

[0026] Figure 1 It can be seen that HSTM-SOD with the amino acid sequence shown in SEQ ID NO:1 has the highest enzyme activity.

[0027] Experimental Example 2 Thermostability verification of superoxide dismutase HSTM-SOD with the above five amino acid sequences.

[0028] The residual enzyme activity of HSTM-SOD with five different amino acid sequences was compared after heat treatment in a 95℃ water bath for 60 min. Commercial superoxide dismutase (SOD) was used as a blank control. The thermostability of different types of HSTM-SOD is shown in [reference needed]. Figure 2 , Figure 2 It is evident that all five HSTM-SODs exhibit better thermal stability than commercially available SODs.

[0029] Experimental Example 3 Enzyme activity verification of HSTM-SOD at different temperatures.

[0030] HSTM-SOD-1 was heat-treated in water baths at different temperatures for 60 min, and its remaining activity was detected. The results are as follows: Figure 3 As shown.

[0031] Figure 3 The results showed that the enzyme exhibited good activity in the temperature range of 0-98℃. After treatment at temperatures below 85℃, the remaining enzyme activity was above 100%. When treated at temperatures above 95℃, the enzyme activity decreased, but still remained above 70%, indicating that the enzyme has excellent thermal stability.

[0032] Test Example 4 HSTM-SOD's ability to cross membranes.

[0033] The transmembrane ability of the obtained HSTM-SOD-1 was investigated, and the experimental results are as follows: Figure 4 As shown.

[0034] Figure 4 As can be seen, the proportion of the target protein HSTM-SOD-1 in the fermentation supernatant is much higher than that in the fermentation cells, indicating that HSTM-SOD-1 has the ability to autonomously cross the cell membrane.

[0035] Experimental Example 5 The residual activity of HSTM-SOD was detected after incubation in buffer solutions of different pH values ​​for 3 h. The results are shown in [Figure number missing]. Figure 5 .

[0036] from Figure 5 The results show that the HSTM-SOD-1 provided by this invention has good pH tolerance, and its enzyme activity remains at 88% or even higher in the pH range of 5.0-10.0.

[0037] Experimental Example 6 This experiment focuses on investigating the fermentation expression level of HSTM-SOD.

[0038] Figure 6 The figure shows the expression levels of the target protein by HSTM-SOD-1 at different time points after induction. As can be seen from the figure, the initial protein expression level can reach more than 30% of the total protein.

[0039] Experimental Example 7 This study focuses on verifying the repair effect of HSTM-SOD on TalaA-induced cell death.

[0040] After inducing cell death with the ferroptosis inducer TalaA, the cells were further treated with two other types of commercially available SOD and the HSTM-SOD prepared in this invention. The experimental results are shown in Table 1 below.

[0041] Table 1. The ability of different HSTM-SODs to rescue ferroptosis. Enzyme class HSTM-SOD-1 HSTM-SOD-2 HSTM-SOD-3 HSTM-SOD-4 HSTM-SOD-5 Iron death rescue ability (%) 67.8 58.1 53.0 59.4 62.2 Enzyme class HSTM-SOD-6 HSTM-SOD-7 HSTM-SOD-8 Control SOD 1 Control SOD 2 Iron death rescue ability (%) 58.9 64.5 57.3 0 2.9

[0042] The results in Table 1 above show that the superoxide dismutases with different sequences provided by this invention have superior ferroptosis rescue capabilities compared to commercially available superoxide dismutases. To more intuitively illustrate the above differences, the anti-ferroptosis capabilities of HSTM-SOD-1, control SOD1, control SOD2, and unadded SOD are plotted as a bar graph. Figure 7 As shown. Figure 7 It is evident that HSTM-SOD-1 can significantly rescue TalaA-induced cell death, demonstrating superior performance compared to other types of SOD.

[0043] Experimental Example 8 This experiment focuses on verifying the ability of HSTM-SOD to repair cells damaged by ultraviolet (UV) radiation.

[0044] Figure 8 This demonstrates the ability of HSTM-SOD-1 to repair UV sunburn. Figure 9 , Figure 10 The images show cell biology experiments and RNA sequencing data, respectively, of the research results on the mechanism of HSTM-SOD-1's ability to repair UV sunburn.

[0045] The above experimental results show that HSTM-SOD-1 can accelerate the repair of UV damage by promoting the expression of epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF1R), and insulin-like growth factor 2 receptor (IGF2R).

[0046] Experimental Example 9 This trial aims to verify the ability of HSTM-SOD to promote wound healing in diabetic patients.

[0047] Table 2 below compares the healing-promoting effects of HSTM-SOD with different sequences in this invention and commercially available ordinary SOD on diabetic wounds.

[0048] Table 2. Healing-promoting effect of different HSTM-SODs on diabetic wounds Enzyme class No SOD HSTM-SOD-1 HSTM-SOD-2 HSTM-SOD-3 HSTM-SOD-4 Wound area (mm 2 )]> 78.5 28.3 32.5 42.5 39.2 Wound healing promotion ability (%) —— 63.9 58.5 45.8 50.1 Enzyme class HSTM-SOD-5 HSTM-SOD-6 HSTM-SOD-7 HSTM-SOD-8 Commercial SOD Wound area (mm 2 )]> 35.3 32.8 29.2 35.7 72.3 Wound healing promotion ability (%) 55.0 58.2 62.8 54.5 7.9

[0049] The experimental data in Table 2 show that the HSTM-SOD provided by this invention can significantly accelerate the healing rate of diabetic wounds. Although the healing ability of different sequences varies, their effects are significantly better than other types of SOD.

[0050] Figure 11The figure shows the healing ability of HSTM-SOD-1 on diabetic wounds, indicating that the skin wounds showed significant healing after treatment with HSTM-SOD-1.

[0051] Experimental Example 10 This study aims to verify the rescue effect of HSTM-SOD-1 on age-related macular degeneration.

[0052] Figure 12 The study demonstrated the rescue effect of different concentrations of HSTM-SOD-1 on age-related macular degeneration (AMD).

[0053] Damage to retinal pigment epithelial (RPE) cells is the currently recognized cellular basis of AMD. When the production and neutralization of reactive oxygen species (ROS) are imbalanced, cells can produce oxidative stress, causing damage to RPE cells and promoting the pathological progression of AMD. In AMD patients, the SOD immune response activity in the cytoplasm and lysosomes of macular RPE cells increases with age, while the SOD activity in peripheral RPE cells decreases with age. Pyrogallol is an oxidative stress modeler that can induce oxidative damage in RPE cells. Figure 12 The results showed that HSTM-SOD at concentrations of 50-200 U / mL was non-toxic to RPE cells themselves, but could rescue RPE cell damage caused by pyrogallol in a concentration-dependent manner, thereby combating AMD.

Claims

1. Superoxide dismutase HSTM-SOD, characterized in that, The amino acid sequence of the superoxide dismutase HSTM-SOD is selected from at least one of the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or a sequence having greater than 80% homology with the above amino acid sequences.

2. The superoxide dismutase HSTM-SOD as described in claim 1, characterized in that, The solution of the superoxide dismutase HSTM-SOD after dissolution is red to reddish-brown in appearance.

3. The gene encoding the superoxide dismutase HSTM-SOD as described in claim 1.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the gene as described in claim 3.

5. A composition for cell protection and / or cell damage repair, characterized in that, The composition contains superoxide dismutase HSTM-SOD as described in any one of claims 1-2 or the recombinant expression vector as described in claim 4.

6. The use of the superoxide dismutase HSTM-SOD of any one of claims 1-2 or the recombinant expression vector of claim 4 in the preparation of products for combating cell ferroptosis-related diseases.

7. The application as described in claim 6, characterized in that, The product described is either a hair loss product or a hair follicle necrosis product that prevents hair loss caused by iron death of hair follicle cells.

8. The use of the superoxide dismutase HSTM-SOD according to any one of claims 1-2 or the recombinant expression vector according to claim 4 in the preparation of products hydrolyzed by resistant digestive enzymes.

9. The use of the superoxide dismutase HSTM-SOD according to any one of claims 1-2 or the recombinant expression vector according to claim 4 in the preparation of a product that promotes wound healing in diabetic patients.

10. The use of the superoxide dismutase HSTM-SOD according to any one of claims 1-2 or the recombinant expression vector according to claim 4 in the preparation of products that alleviate oxidative damage to fundus cells.

Images