Fusion protein containing SUMO tag and preparation method and application thereof

Abstract

The invention relates to the technical field of biology, and particularly discloses a fusion protein containing an SUMO tag and a preparation method and application thereof. The fusion protein provided by the invention comprises an SUMO tag and an antibacterial peptide, the antibacterial peptide can inhibit escherichia coli and staphylococcus aureus, and the amino acid sequence of the antibacterial peptide is as shown in SEQ ID No. 10, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 12, SEQ ID No. 15 or SEQ ID No. 17. The fusion protein can be efficiently expressed through a prokaryotic expression system, renaturation of the antibacterial peptide can be successfully achieved after enzyme digestion and purification, the antibacterial activity of the antibacterial peptide is recovered, and therefore efficient preparation of the antibacterial peptide is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a fusion protein containing a SUMO tag and a preparation method and application thereof. Background Art

[0002] With the long-term and extensive use of antibiotics, bacteria and other pathogens have become increasingly resistant to antibiotics, resulting in a decline in the effectiveness of traditional antibiotic treatments, and even a situation where no drugs are available, which seriously threatens human health. Therefore, it is necessary to find other alternatives to resist pathogens.

[0003] Antimicrobial peptides are a class of small molecule polypeptides widely found in organisms in nature, usually composed of 12-50 amino acid residues. They are an important component of the innate immune system in organisms, have broad-spectrum antimicrobial activity, and can fight against a variety of pathogens such as bacteria, fungi, viruses, and parasites. And because of their unique antimicrobial mechanism, pathogens are not easy to develop resistance to them, so they are considered to be a strong candidate for solving the problem of antibiotic resistance.

[0004] There are several main methods for preparing antimicrobial peptides: 1. Direct extraction method: extract antimicrobial peptides directly from organisms. This method is simple but has low yield and is suitable for laboratory research. 2. Enzymatic hydrolysis: obtain antimicrobial peptides by enzymatic hydrolysis of proteins. This method uses specific enzymes to break down proteins into small peptide fragments, but requires complex separation and screening processes, which is not conducive to the production of large-scale industrial pure products. 3. Chemical synthesis method: The main methods include liquid phase synthesis (LPPS) and solid phase synthesis (SPPS). Liquid phase synthesis (LPPS) is a method for synthesizing peptides by directly connecting amino acids in solution. Solid phase synthesis technology (SPPS) is to synthesize peptide chains from C-terminus to N-terminus by repeatedly adding amino acids to a solid support. However, the chemical synthesis method requires precise control of the operation process and is relatively expensive. 4. Recombinant DNA technology: Genetic engineering technology is used to express antimicrobial peptides in microorganisms (such as Escherichia coli and yeast). This method is more suitable for large-scale production of antimicrobial peptides.

[0005] In recombinant DNA technology, the expression of antimicrobial peptides faces a key problem: their own antimicrobial activity may cause damage to host cells. When host cells express antimicrobial peptides, it may trigger the cell's self-protection mechanism, resulting in decreased expression efficiency or cell damage, and even affecting the yield and activity of antimicrobial peptides, thereby increasing production costs.

[0006] To solve this problem, the strategy of co-expression of fusion tags and antimicrobial peptides is usually adopted. However, if the fusion tag is not selected properly, the antimicrobial peptide may be insufficiently expressed or lose its activity in subsequent treatments. Therefore, how to accurately select the appropriate fusion tag to ensure the efficient and safe expression of antimicrobial peptides in host cells, and at the same time, optimize the enzyme cutting, cleavage and renaturation steps to finally obtain high-purity and active antimicrobial peptides, is still an important direction of current research. Summary of the invention

[0007] One of the purposes of the present invention is to provide a fusion protein that can be efficiently expressed in host cells and still has antibacterial activity after expression and renaturation.

[0008] The present invention provides a fusion protein, which comprises: a SUMO tag and an antimicrobial peptide; the antimicrobial peptide can inhibit Escherichia coli and Staphylococcus aureus, and the amino acid sequence of the antimicrobial peptide is shown in SEQ ID No.10, SEQ ID No.6, SEQ ID No.8, SEQ ID No.12, SEQ ID No.15 or SEQ ID No.17.

[0009] In genetic recombination engineering, the SUMO (Small Ubiquitin-related Modifier) ​​tag is a fusion tag. It is a small protein tag derived from the SUMO protein family. SUMO protein exists naturally in eukaryotic cells and plays an important role in many cellular processes, such as protein localization, stability and functional regulation. However, not all antimicrobial peptides can be successfully expressed using this tag.

[0010] The present invention has found through a large number of experiments that only when a specific fusion tag and an antimicrobial peptide are combined can the antimicrobial activity of the antimicrobial peptide after renaturation be retained while ensuring the effective expression of the fusion protein. However, there is no universal rule to follow for different antimicrobial peptides / fusion tags as to what specific combination method can achieve the expected results. The inventor needs to conduct a large number of experiments to explore in order to obtain a fusion protein with a good combination effect.

[0011] The fusion protein of the present invention is obtained through a large number of exploratory experiments. Specifically, the present invention combines a specific fusion tag with an antimicrobial peptide to successfully achieve soluble expression of the fusion protein. This fusion protein does not have the antimicrobial activity of the original antimicrobial peptide, thus avoiding damage to the host cells. The separate antimicrobial peptide released after purification and lysis still has a good antibacterial effect, thereby realizing the large-scale preparation of the specific antimicrobial peptide of the present invention in a prokaryotic expression system.

[0012] In the fusion protein of the present invention, the amino acid sequence of the SUMO tag is shown as SEQ ID No.1.

[0013] The antimicrobial peptides of the present invention can form soluble co-expression with SUMO.

[0014] In the fusion protein of the present invention, a ULP1 protease cleavage site is also included between the SUMO tag and the antimicrobial peptide.

[0015] The amino acid sequence of the fusion protein of the present invention is shown as SEQ ID No.23, SEQ ID No.19, SEQ ID No.21, SEQ ID No.25, SEQ ID No.27 or SEQ ID No.29.

[0016] The present invention also provides a nucleic acid encoding the above fusion protein.

[0017] The sequence of the nucleic acid of the present invention is shown as SEQ ID No.24, SEQ ID No.20, SEQ ID No.22, SEQ ID No.26, SEQ ID No.28 or SEQ ID No.30.

[0018] The present invention also provides a biological material containing the above nucleic acid, wherein the biological material is an expression cassette, a vector or a host cell.

[0019] The present invention also provides the use of the above fusion protein, nucleic acid or biological material in the preparation of antimicrobial peptides.

[0020] The present invention also provides a method for preparing antimicrobial peptides, which comprises the step of causing Escherichia coli to express the fusion protein.

[0021] The method of the present invention comprises: (1) inserting the above nucleic acid into an expression vector to obtain an expression plasmid; (2) transforming the expression plasmid into Escherichia coli; (3) expanding the culture of the Escherichia coli transformed with the expression plasmid and inducing expression; In the induced expression, the temperature of the induced expression is 16°C-37°C, preferably 32°C; Induce expression with IPTG at a final concentration of 0.25mM-2.0mM, preferably induce expression with IPTG at a final concentration of 0.75mM-1.0mM; The induction expression time is 1-6 hours, preferably 5-6 hours.

[0022] With the preferred induction expression conditions of the present invention, a better expression amount can be obtained.

[0023] The beneficial effects of the present invention are at least: The fusion protein of the present invention can inhibit the antibacterial effect of the antimicrobial peptide on Escherichia coli and avoid causing damage to the host cells, thereby achieving efficient expression through a prokaryotic expression system. After the fusion protein is expressed, the renaturation of the antimicrobial peptide can be successfully achieved through enzyme cleavage, thereby restoring the inhibitory activity of the antimicrobial peptide on Escherichia coli, thereby achieving efficient preparation of the antimicrobial peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the map of SUMO+ S4 / S5 / S6 / S7 / S10 / S12 in the pET-28a expression vector.

[0025] Figure 2 These are the gel electrophoresis results of the antimicrobial peptide fusion protein SUMO+QIGG +S4, S5, S6, S7, S10, and S12 expressing soluble fusion proteins.

[0026] Figure 3 The results show the effect of induction temperature on the culture of S6 fusion protein. Lanes 1-5 correspond to the results of induction temperatures of 16°C, 24°C, 28°C, 32°C, and 37°C, respectively.

[0027] Figure 4 The results show the effect of IPTG concentration on the culture of S6 fusion protein. Lanes 1-5 correspond to the results of IPTG concentrations of 0.25mM, 0.75mM, 1.0mM, 1.5mM, and 2.0mM, respectively.

[0028] Figure 5 The results of the effects of different induction times on the culture of S6 fusion protein. Lanes 1-6 correspond to the results of induction time of 1h, 2h, 3h, 4h, 5h, and 6h, respectively.

[0029] Figure 6 These are the antibacterial test results of the antimicrobial peptides S4, S5, S6, S7, S10, and S12 prepared in the examples.

[0030] Figure 7 These are the plate antibacterial test results of the S4, S5, S6, S7, S10, and S12 antimicrobial peptides prepared in the examples.

[0031] Figure 8 The gel electrophoresis results of the comparative antimicrobial peptide fusion protein SUMO+QIGG+S1, S2, S3, S8 expressing soluble fusion proteins.

[0032] Fig. 9 The antibacterial test results of S1, S2, S3 and S8 antimicrobial peptides prepared in comparative examples are shown. DETAILED DESCRIPTION

[0033] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0034] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0035] Example 1-1 Preparation of antimicrobial peptides This example provides a method for expressing an antimicrobial peptide having an antibacterial effect on Escherichia coli. Specifically, the preparation of the antimicrobial peptide is achieved by expressing an antimicrobial peptide fusion protein containing a SUMO tag. The method is as follows: 1. Preparation of SUMO+QIGG+antimicrobial peptide fusion protein containing purified cleavage site: The sequences of the fusion proteins are: SUMO +QIGG+ S4, SUMO +QIGG+ S5, SUMO +QIGG+ S6, SUMO +QIGG+ S7, SUMO +QIGG+ S10, SUMO +QIGG+ S12, which are used for prokaryotic expression of S4, S5, S6, S7, S10, and S12 antimicrobial peptides, respectively.

[0036] The amino acid sequences of the fusion proteins SUMO +QIGG+ S4, SUMO +QIGG+ S5, SUMO +QIGG+ S6, SUMO +QIGG+ S7, SUMO +QIGG+ S10, and SUMO +QIGG+ S12 are shown in SEQ ID No.19 (corresponding to S4), SEQ ID No.21 (corresponding to S5), SEQ ID No.23 (corresponding to S6), SEQ ID No.25 (corresponding to S7), SEQ ID No.27 (corresponding to S10), and SEQ ID No.29 (corresponding to S12), and the corresponding base sequences are shown in SEQ ID No.20, SEQ ID No.22, SEQ ID No.24, SEQ ID No.26, SEQ ID No.28, and SEQ ID No.30. Each fusion protein contains a SUMO tag sequence and a restriction site QIGG sequence for UPL1 protease cleavage.

[0037] Specifically, the base sequences of SUMO +QIGG+ S4, SUMO +QIGG+ S5, SUMO +QIGG+ S6, SUMO +QIGG+ S7, SUMO +QIGG+ S10, and SUMO +QIGG+ S12 used in this example were commissioned to Shanghai Huajin Biotechnology Co., Ltd. to synthesize the gene fragments, and the synthesized gene fragments were inserted into the PET-28a expression vector through the restriction sites of BamH I (NEB Company, Catalog Number: R0136L) and Xho I (NEB Company, Catalog Number: R0146L), respectively. The schematic diagram is shown in Figure 1 .

[0038] The amino acid sequence of the SUMO tag is shown in SEQ ID No. 1, and the codon-optimized base sequence thereof for E. coli expression is shown in SEQ ID No. 2.

[0039] The amino acid sequences of the antimicrobial peptides S4, S5, S6, S7, S10 and S12 are shown in SEQ ID No.6 (S4), SEQ ID No.8 (S5), SEQ ID No.10 (S6), SEQ ID No.12 (S7), SEQ ID No.15 (S10) and SEQ ID No.17 (S12), respectively, and the corresponding base sequences are shown in SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13, SEQ ID No.16 and SEQ ID No.18, respectively.

[0040] In this example, the six expression plasmids constructed successfully above were transformed into Escherichia coli competent cells BL21 (DE3) (Merck). The specific process is as follows: (1) Place 1 µl of the plasmid in 100 µl of competent E. coli BL21 (DE3) cells and place on ice for 30 min.

[0041] (2) The mixture was heat-shocked in a 42°C water bath for 90 seconds and then quickly placed on ice for 2 minutes.

[0042] (3) Add 600 µl of resistance-free LB to the mixture and incubate at 37°C, 220 rpm for 1 h.

[0043] (4) Take 200 µl of the bacterial solution and spread it evenly on an LB plate containing kanamycin sulfate resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 100 µg / ml kanamycin).

[0044] (5) Place the plate upside down in a 37°C incubator for about 20 hours until clearly visible colonies appear.

[0045] In this example, a monoclonal colony was picked from the transformed LB plate and cultured in 10 ml LB (containing 100 μg / ml kanamycin) medium for 12 hours, and then transferred to 2×YT medium (16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride) at a ratio of 1:100 for expansion culture, and cultured at 37°C and 220 rpm until the bacterial solution OD600 was 0.4-0.6, and IPTG (Sigma, catalog number: I5502-1G) with a final concentration of 0.25 mM was added to induce expression, and the induction conditions were 16°C and 180 rpm for 4 hours. Finally, the bacteria were collected by centrifugation and stored at -20°C or immediately entered the next step of purification.

[0046] In this example, about 50 ml of phosphate buffer (pH 7.8) (40 mM sodium dihydrogen phosphate, 500 mM sodium chloride) was used to resuspend (1 L) the bacterial precipitate, and the bacteria were lysed using a high-pressure lysis instrument (Xinzhi Biotechnology). The precipitate was centrifuged at 13,000 rpm for 30 min, and the soluble protein in the supernatant was collected.

[0047] In this example, the fusion protein was purified using a Ni column. The Ni-NTA (Qiagen, Cat. No. 30210) affinity column was balanced with a binding buffer (40mM NaH2PO3, 150mM NaCl, pH7.8). Then, the soluble protein supernatant was added and incubated at 4°C for 0.5-1h to allow the fusion protein to fully bind to the column material. Then, the impurities were rinsed with 200ml of a washing buffer (10mM imidazole, 40mM NaH2PO3, 150mM NaCl, pH 7.8) containing 10mM imidazole (Sigma). Finally, 20ml of an elution buffer (250mM imidazole, 40mM NaH2PO3, 150mM NaCl, pH 7.8) containing 250mM imidazole was added to elute the fusion protein. The resulting product can be analyzed for concentration and purity by SDS-PAGE electrophoresis. The specific process is: take 40μl of protein solution, add 10μl 5× protein loading buffer (250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), boil in 100℃ boiling water for 10min, then add 10μl per well to SDS-PAGE protein gel, run at 80V for 2h, use Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 20min, and then use protein decolorizing solution (10% acetic acid, 5% ethanol) for decolorization. The gel electrophoresis results of this example are shown in Figure 2(Two replicates for each fusion protein), which showed that the antimicrobial peptide fusion protein was expressed correctly.

[0048] 2. Preparation of antimicrobial peptides S4, S5, S6, S7, S10 and S12: In this example, the fusion protein was cleaved with ULP1 protease to release free S4, S5, S6, S7, S10, and S12 antimicrobial peptides. ULP1 protease was added to the fusion protein solution at a volume ratio of 50:1, and the fusion protein solution was cleaved overnight at room temperature to release S4, S5, S6, S7, S10, and S12 antimicrobial peptides.

[0049] The S4, S5, S6, S7, S10, and S12 antimicrobial peptides released in this embodiment can be purified by dialysis, and then tested for activity or freeze-dried for later use. The specific process is: transfer the S4, S5, S6, S7, S10, and S12 antimicrobial peptides to a dialysis bag with a molecular weight cutoff of 1 kDa, place them in a distilled water solution with a volume of 100 times the dialysate, dialyze for 24 hours in a 4°C environment, continue stirring, and change the distilled water every 4 hours. Then collect the solution in the dialysis bag, take the supernatant after centrifugation, and obtain the purified S4, S5, S6, S7, S10, and S12 antimicrobial peptides.

[0050] Example 1-2 Experimental Condition Optimization In this example, the induction culture conditions of the S6 antimicrobial peptide host cells were screened. Specifically, the Escherichia coli transformed with the expression plasmid was transferred to 2×YT medium (16g / L peptone, 10g / L yeast extract, 5g / L sodium chloride) at a ratio of 1:100 for expansion culture. The culture was cultured at 37°C and 220rpm until the OD600 of the bacterial solution was 0.4-0.6. The effects of different induction temperatures, different concentrations of IPTG, and different induction times on the expression results of the fusion protein were investigated. For specific induction expression methods, see Example 1-1. First, the induction temperatures were set to: 16°C, 24°C, 28°C, 32°C, and 37°C. After the fusion proteins were prepared, the induction results were tested. The electrophoresis diagram of the fusion protein SUMO +QIGG+ S6 is shown in Figure 3 It can be seen that with the increase of induction temperature, the expression level of the SUMO-tagged fusion protein targeting S6 showed a trend of first increasing and then decreasing; the expression level was the highest at 32°C.

[0051] Continue to set the condition difference and set the IPTG concentration to: 0.25mM, 0.75mM, 1.0mM, 1.5mM, 2.0mM. When the final IPTG concentration is 0.75mM-1.0mM, the expression level of S6 fusion protein is the highest. The results are shown in Figure 4 .

[0052] Continue to set the induction time to: 1h, 2h, 3h, 4h, 5h, 6h. As the induction time increases, the expression level of the S6 antimicrobial peptide fusion protein increases with the increase of the induction time. The expression level is the highest at 5-6h. For the results, see Figure 5 .

[0053] Example 1-3 Antibacterial Experiment This example tests the antibacterial activity of the antimicrobial peptide obtained in Example 1-1, as follows: 1. Antibacterial experiment: (1) Thaw the frozen Escherichia coli and Staphylococcus aureus strains separately and inoculate them into 100 μl solid culture medium. Pick a single clone at 37 degrees overnight. Select a single clone colony and inoculate it into a 20 ml liquid culture medium (LB) flask. Place it on a shaker at 180 rpm and incubate it at 37 degrees for 16 hours as a bacterial stock solution.

[0054] (2) Blank control group: Take 1 ml of bacterial stock solution, add 5 ml of liquid culture medium LB, mix well, culture at 37 degrees for 4 hours on a shaker, and measure the OD value at 600 nm.

[0055] (3) Antimicrobial peptide experimental group: 1 ml of bacterial stock solution was taken, and the corresponding antimicrobial peptide was added at a final concentration of 50 ug / ml. The mixture was mixed evenly, and cultured on a shaker at 37°C for 4 h. The OD value at 600 nm was measured.

[0056] (4) Antibiotic control group: 1 ml of bacterial stock solution was taken, and antibiotics were added at a final concentration of 50 μg / ml. The mixture was mixed well, and cultured on a shaker at 37°C for 4 h. The OD value at 600 nm was measured.

[0057] OD value results are shown in Figure 6 The inhibition rate was further calculated based on the OD value results.

[0058] Inhibition rate = (OD value of bacterial solution in the blank group - OD value of bacterial solution in the experimental group) / OD value of bacterial solution in the blank group × 100%.

[0059] 2. Plate antibacterial experiment: According to the E. coli culture, several plates were laid out and lines were drawn to distinguish the sample positions; water was dripped into the blank group, antimicrobial peptide was dripped into the experimental group, and kanamycin was dripped into the control group, and cultured overnight. Whether the antimicrobial peptide showed empty plaque phenomenon was observed to determine the antibacterial activity. The results are shown in Figure 7 .

[0060] according to Figure 6The antibacterial test results of the antimicrobial peptides S4, S5, S6, S7, S10, and S12 prepared in Example 1-1 (Escherichia coli and Staphylococcus aureus, respectively, and the control group was kanamycin) show that the inhibitory effects of the six antimicrobial peptides are as follows: at a concentration of 50ug / ml, the inhibition rate of antimicrobial peptide S4 against Escherichia coli is 83.50%, the inhibition rate of antimicrobial peptide S5 is 95.38%, the inhibition rate of antimicrobial peptide S6 is 100%, the inhibition rate of antimicrobial peptide S7 is 89.20%, the inhibition rate of antimicrobial peptide S10 is 93.80%, and the inhibition rate of antimicrobial peptide S12 is 97.61%. %; At a concentration of 50ug / ml, the inhibition rates of antimicrobial peptide S4, S5, S6, S7, S10, S12, S10% and S10% against Staphylococcus aureus were 93.23%, 96.10%, 97.56%, 94.89%, 97.16% and 100% respectively.

[0061] according to Figure 7 From the plate antibacterial test of the S4, S5, S6, S7, S10 and S12 antimicrobial peptides prepared in the examples, it can be seen that against Escherichia coli, the S4, S5, S6, S7, S10 and S12 antimicrobial peptides all showed plaque phenomenon, which is similar to the effect of kanamycin.

[0062] Comparative Example In this comparative example, the SUMO tag was fused with the antimicrobial peptides S1, S2, S3, and S8 according to the method described in Example 1-1, wherein the amino acid sequences of S1, S2, S3, and S8 are SEQ ID No.3 (S1), SEQ ID No.4 (S2), SEQ ID No.5 (S3), and SEQ ID No.14 (S8), respectively. However, the fusion proteins S2 and S3 antimicrobial peptides were not expressed. Figure 8 After purification, the S1 and S8 antimicrobial peptides have no antibacterial activity (see Examples 1-3 for the test method). Fig. 9 Therefore, not all antimicrobial peptides can be expressed using the SUMO tag.

[0063] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A fusion protein, characterized in that include: SUMO tag and antimicrobial peptides; The antimicrobial peptide can inhibit Escherichia coli and Staphylococcus aureus, and the amino acid sequence of the antimicrobial peptide is shown as SEQ ID No.10, SEQ ID No.6, SEQ ID No.8, SEQ ID No.12, SEQ ID No.15 or SEQ ID No.

17.

2. The fusion protein according to claim 1, characterized in that The amino acid sequence of the SUMO tag is shown in SEQ ID No.

1.

3. The fusion protein according to claim 1 or 2, characterized in that A ULP1 protease cleavage site is also included between the SUMO tag and the antimicrobial peptide.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID No.23, SEQ ID No.19, SEQ ID No.21, SEQ ID No.25, SEQ ID No.27 or SEQ ID No.

29.

5. A nucleic acid encoding the fusion protein according to any one of claims 1 to 4.

6. The nucleic acid according to claim 5, characterized in that The sequence is shown as SEQ ID No.24, SEQ ID No.20, SEQ ID No.22, SEQ ID No.26, SEQ ID No.28 or SEQ ID No.

30.

7. A biological material containing the nucleic acid according to claim 5 or 6, wherein the biological material is an expression cassette, a vector or a host cell.

8. Use of the fusion protein according to any one of claims 1 to 4, or the nucleic acid according to claim 5 or 6, or the biomaterial according to claim 7 in the preparation of antimicrobial peptides.

9. A method for preparing an antimicrobial peptide, characterized in that: The method comprises the step of causing Escherichia coli to express the fusion protein according to any one of claims 1 to 4.

10. The method according to claim 9, characterized in that include: (1) inserting the nucleic acid according to claim 5 or 6 into an expression vector to obtain an expression plasmid; (2) transforming the expression plasmid into Escherichia coli; (3) expanding the culture of the Escherichia coli transformed with the expression plasmid and inducing expression; In the induced expression, the temperature of the induced expression is 16°C-37°C, preferably 32°C; Induce expression with IPTG at a final concentration of 0.25mM-2.0mM, preferably with IPTG at a final concentration of 0.75mM-1.0mM; The induction expression time is 1-6 hours, preferably 5-6 hours.

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