A fibroblast growth factor 21 - penetratin fusion protein, its preparation method and applications

Abstract

The present invention discloses a fibroblast growth factor 21 - penetratin fusion protein, a preparation method thereof and an application thereof. The amino acid sequence of the protein is shown in SEQ ID NO.1, and a preparation method for producing a protein with high purity by Escherichia coli expression is further provided. The FGF21 fused with TP1 provided by the present invention can improve the biological safety and stability of FGF21, does not affect the biological activity of FGF21, and solves the problem of low purity of FGF21 after purification in the Escherichia coli expression system, and can significantly improve the protein purification yield of FGF21 produced by Escherichia coli expression, greatly improving the application prospect of protecting the skin from photo - damage; this provides a new application prospect for FGF21 as a new skin photo - damage protectant.

Description

Technical Field

[0001] The present invention relates to a fusion protein, and particularly to a fibroblast growth factor 21 - penetratin fusion protein, a preparation method thereof and an application thereof. Background Art

[0002] Fibroblast growth factor 21 (FGF21) is an important member of the fibroblast growth factor (FGFs) family. The human FGF21 gene is located on chromosome 19 and can encode 209 amino acids. After the protein matures, the signal peptide at the amino - terminal can be degraded into 181 amino acids. There is a 75% amino acid homology sequence between human and mouse FGF21. FGF21 can not only reduce serum cholesterol, triglyceride (TG) and plasma glucose, but also improve insulin sensitivity. In addition, FGF21 can protect the skin from ultraviolet damage. Therefore, FGF21 has great potential for developing sunscreen skin care products.

[0003] However, the effect of protecting against ultraviolet damage by FGF21 has not been obvious at present, which limits its application in skin sunscreen. The fusion protein strategy provides an effective solution to improve the protective effect of FGF21 against ultraviolet damage. There is a study that constructs a recombinant protein DF2 - HSA by combining human β - defensin 2 and human serum albumin HSA. This recombinant fusion protein has stronger killing power against tumor cells than free defensin HBD2 and HSA, and the recombinant protein DF2 - HSA can quickly aggregate at the tumor site and stay for a longer time, while free defensin HBD2 is quickly metabolized. Therefore, it can be seen that fusion proteins may be able to improve some main properties of proteins. Summary of the Invention

[0004] The purpose of the present invention is to overcome the disadvantages and deficiencies existing in the prior art, and to provide a fibroblast growth factor 21 - penetratin fusion protein, a preparation method thereof and an application thereof.

[0005] The technical solution adopted by the present invention is as follows: A fibroblast growth factor 21 - penetratin fusion protein, and the amino acid sequence of the protein is as shown in SEQ ID NO. 1.

[0006] A gene encoding the above - mentioned fusion protein, and the nucleotide sequence of the gene is as shown in SEQ ID NO. 2.

[0007] A recombinant vector containing the above - mentioned gene.

[0008] The above - mentioned recombinant vector is prepared by pET15b.

[0009] A recombinant bacterium containing the above - mentioned recombinant vector.

[0010] Use of the fusion protein as described above in the preparation of a skin photo-damage protectant.

[0011] A preparation method for expressing the fusion protein as described above in an E. coli system, the method comprising the following steps:

[0012] (1) Use Nde I and Kpn I restriction endonucleases to cut down the TP-1 gene on the vector, use Kpn I and BamH I restriction endonucleases to cut down the FGF21 gene on the vector, use Nde I and BamH I restriction endonucleases to cut open the pET15b vector, use a DNA recovery kit to recover the cut TP-1 gene, FGF21 gene and the cut pET15b vector, ligate with T4 ligase, transform the ligation product into E. coli, screen out the gene encoding the fusion protein as described above, name this gene pET15b-TP1-FGF21, and extract the pET15b-TP1-FGF21 vector plasmid;

[0013] (2) Transfer the extracted pET15b-TP1-FGF21 vector plasmid into ARC-BL21(DE3) E. coli competent cells by heat shock method, and use PCR method to screen out and identify the E. coli containing the pET15b-TP1-FGF21 plasmid; place the identified E. coli in a shaker at 37 °C and 200 rpm for culture until the absorbance OD 600 of the E. coli broth reaches 1.0, and use centrifugation to collect 1 ml of the cultured E. coli to prepare a sample before induction;

[0014] (3) When OD 600 reaches 1.0, add the inducer IPTG with a final concentration of 50 mM, continue to culture at 37 °C and 200 rpm, and when OD 600 reaches 2.5, use centrifugation to collect the cultured E. coli, and take another 1 ml of the induced E. coli broth to prepare an electrophoresis sample.

[0015] In step (2), screening out and identifying the E. coli containing the pET15b-TP1-FGF21 plasmid by PCR method includes the following process:

[0016] Use a genomic DNA extraction kit to extract the whole genome of the E. coli into which the pET15b-TP1-FGF21 plasmid has been introduced, use PCR method to separate the amplification product through agarose gel with upstream and downstream primers, and detect it with a UV imager;

[0017] Culture the E. coli with positive PCR identification until OD 600 reaches 1.0, take 1 ml of the bacterial liquid as a sample before induction, and add the inducer IPTG with a final concentration of 50 mM; continue to culture for 6 hours until OD 600When it reached 2.5, 1 mL of the induced Escherichia coli bacterial solution was taken for centrifugation, the loading buffer was added, and it was subjected to a boiling water bath. The extracted Escherichia coli protein was separated by SDS-PAGE gel and transferred to a PVDF membrane using a membrane transfer instrument. The blocking solution was added and blocked at room temperature. After washing off the blocking solution, anti-rabbit FGF21 antibody was added and incubated overnight at 4°C; after washing off the antibody, alkaline phosphatase-labeled rabbit anti-mouse antibody was added and incubated at room temperature; after washing off the rabbit anti-mouse antibody, the chromogenic agent BCIP / NBT was added and placed in an imager for detection; through molecular biology detection, strains of transformed pET15b-TP1-FGF21 Escherichia coli that highly expressed the above-mentioned fusion protein were screened out.

[0018] The beneficial effects of the present invention are as follows:

[0019] 1. The fibroblast growth factor 21-transdermal peptide fusion protein provided by the present invention can improve the biological safety and stability of FGF21, does not affect the biological activity of FGF21, and FGF21 fused with TP1 significantly improves the efficacy of FGF21 and greatly improves its application prospect of reducing the damage of ultraviolet rays to the skin; at the same time, transdermal drug delivery can also reduce the pain caused by microneedles;

[0020] 2. The Escherichia coli expression system has the advantages of fast reproduction, low cost, high expression level, easy purification of the expression product, good stability, etc. The fusion protein of the present invention is produced by Escherichia coli expression without affecting its biological activity. This fusion protein production system has no potential risks and reduces the harm to the skin;

[0021] 3. The fusion protein production system of the present invention is simple to prepare, has obvious transdermal effects, and from the results of cell experiments, when TP1 and FGF21 are fused and expressed, it has no effect on their respective structures and has no adverse effects on the tested cells. Therefore, this fusion protein belongs to a biosafe substance.

[0022] The present invention fuses TP1 in order to improve the effect of FGF21 in protecting against ultraviolet damage. The preparation of the fusion protein has become a key issue for whether FGF21 can be applied to sunscreen skin care. In order not to affect the function of the fusion protein, the present invention adds a flexible linker (GGGGT) between the two functional proteins. Since the prokaryotic expression system has the advantages of being able to obtain a brand-new gene product in a short period, the target product has a stable and consistent structure, the experimental method is easy to explore and optimize, and the target product is easy to separate and purify, the present invention attempts to use the prokaryotic expression system to prepare the fusion protein. Experimentally, the expression conditions are improved, such as adjusting the concentration of the inducer IPTG, adjusting the expression time of the bacterial strain, and replacing the protein purification eluent, etc. Through the above methods, high-purity FGF21 fusion protein can be effectively prepared without changing its biological activity. Thus, FGF21 can be applied to new sunscreen skin care products at an early date. Description of the Drawings

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, obtaining other accompanying drawings based on these drawings still belongs to the scope of the present invention.

[0024] Figure 1 It is a process diagram of the transformation of pET15b-TP1-FGF21 mediated by Escherichia coli of the present invention;

[0025] Figure 2 It is a PCR detection diagram of the TP1-FGF21 plasmid system of the present invention;

[0026] Figure 3 It is a Western-blot detection diagram of the TP1-FGF21 Escherichia coli system of the present invention;

[0027] Figure 4 It is a protein purification result diagram of TP1-FGF21 of the present invention;

[0028] Figure 5 It is a detection diagram of the effect of TP1-FGF21 of the present invention on DNA damage of HaCaT cells induced by UVB;

[0029] Figure 6 It is a detection diagram of the effect of TP1-FGF21 of the present invention on the mitochondrial membrane potential of UVB-damaged HaCaT cells. Detailed implementation manners

[0030] To make the objectives, technical solutions and advantages of the present invention clearer, the following will further describe the present invention in detail with reference to the accompanying drawings.

[0031] Example 1

[0032] A preparation method of a vector of fibroblast growth factor fused with a transdermal peptide, the method comprising:

[0033] (1) Construction of a fusion gene expression vector of TP-1 and FGF21

[0034] Using Nde I and Kpn I restriction enzymes to cut out the TP-1 gene stored in the cloning vector in our laboratory, using Kpn I and BamH I restriction enzymes to cut out the FGF21 gene stored in the cloning vector in our laboratory, using Nde I and BamHThe pET15b vector was cut by I endonuclease, and the excised TP-1 gene fragment, FGF21 gene fragment and the excised pET15b vector were mixed in proportion, the DNA fragments were ligated with T4 ligase, the ligation product was transformed into Escherichia coli, the clone bacteria containing the desired vector were screened out, and the desired vector was named pET15b-TP1-FGF21. As Figure 1 shown, this is the construction diagram of the pET15b-TP1-FGF21 expression vector of the present invention.

[0035] (2) Transformation of pET15b-TP1-FGF21 plasmid into Escherichia coli competent cells

[0036] The plasmid of pET15b-TP1-FGF21 vector was extracted, and the extracted plasmid was transferred into ARC-BL21(DE3) Escherichia coli competent cells by heat shock method. Escherichia coli containing pET15b-TP1-FGF21 plasmid was screened by PCR method. The upstream primer sequence of the upstream and downstream primers used was as shown in SEQ ID NO. 3, and the downstream primer sequence was as shown in SEQ ID NO. 4. The identified Escherichia coli was placed in a 50 ml culture flask and cultured in a shaker at a temperature of 37 °C and a rotation speed of 200 rpm for 4 hours to make the absorbance OD of the Escherichia coli broth 600 reach 1.0. 1 ml of the broth was collected as a sample before induction, and then IPTG with a final concentration of 50 mM was added to induce the expression of the fusion protein, and it was continued to be cultured in the shaker for 6 h. When the absorbance OD of the Escherichia coli broth 600 reached 2.5, the cultured Escherichia coli was collected by centrifugation, and the precipitate after centrifugation was the Escherichia coli cells.

[0037] As Figure 2 shown, this is the TP1-FGF21 Escherichia coli transformation process diagram. From left to right are protein Marker, Escherichia coli before induction, and Escherichia coli after induction. It can be seen from the figure that after adding the inducer, at 25KD, compared with before induction, there is an obvious band of the TP1-FGF21 target protein after induction, indicating that the plasmid carrying the TP1-FGF21 target gene has been successfully transformed into Escherichia coli.

[0038] (3) Screening of the Escherichia coli expression system containing the TP1-FGF21 fusion protein

[0039] Extract the whole genome of TP1-FGF21 Escherichia coli using a genomic DNA extraction kit. Run the program in a PCR instrument with the upstream and downstream primers synthesized by the company: 95°C for 5 min; (95°C for 30 sec; 60°C for 30 sec; 72°C for 70 sec) × 30 cycles; 72°C for 8 min. Separate the amplified products by agarose gel electrophoresis and detect them using a UV imager. The sequence of the upstream primer among the upstream and downstream primers used is shown in SEQ ID NO. 3, and the sequence of the downstream primer is shown in SEQ ID NO. 4.

[0040] Pick single colonies of the transformed Escherichia coli and streak them on another plate for culturing. After culturing in an incubator for 4 hours, pick the streaked part of the bacteria and add it to an LB medium containing ampicillin antibiotic for enlarged culturing. When the OD 600 reaches 1.0, take 1 ml of the bacterial liquid as the pre-induction sample, then add the inducer IPTG with a final concentration of 50 mM, and continue culturing in a shaker for 6 hours. When the OD 600 reaches 2.5, take 1 ml of the bacterial liquid as the post-induction sample. Then centrifuge the bacterial liquid samples before and after induction of Escherichia coli. The precipitate obtained is the fusion protein. Add protein loading buffer (the volume ratio of the mixture to protein loading buffer is 10∶1), and perform a boiling water bath for 10 min. Separate the extracted TP1-FGF21 fusion protein by SDS-PAGE gel. Add a part to Coomassie Brilliant Blue staining solution for staining. After staining for 2 hours, suck out the staining solution and add decolorizing solution for decolorization. Place it in an Image Lab imager for detection; transfer another part to a PVDF membrane using a membrane transfer instrument, add blocking solution and block at room temperature for 2 h. Wash off the blocking solution and add rabbit anti-FGF21 antibody diluted 1:1000, and incubate overnight at 4°C; wash off the antibody and add mouse antibody labeled with alkaline phosphatase diluted 1:10000, and incubate at room temperature for 2 h; wash off the mouse antibody and add chromogenic agent BCIP / NBT, and place it in an imager for detection; screen out 2 strains through molecular biology detection, and name them TP1-FGF21-001 and TP1-FGF21-002 respectively.

[0041] As Figure 3 shown, the Western-blot detection diagram of the TP1-FGF21 Escherichia coli system and the PCR detection diagram of the TP1-FGF21 plasmid system of the present invention. A is the Western-blot detection diagram of the TP1-FGF21 Escherichia coli system; B is the PCR detection diagram of the TP1-FGF21 plasmid system. From Figure 3It is known that Figure A is the Western-blot detection diagram of the TP1-FGF21 Escherichia coli expression system. From left to right are Marker, the sample before induction of TP1-FGF21, and the sample after induction of TP1-FGF21. From the results, it can be seen that under the treatment of the inducer IPTG, Escherichia coli can correctly express the TP1-FGF21 recombinant fusion protein; Figure B is the PCR detection diagram of the TP1-FGF21 plasmid system. From left to right are Marker, TP1-FGF21 plasmids 1-7, and the 8th lane is the blank control. From the figure, it can be seen that the TP1-FGF21 plasmid system can be successfully amplified.

[0042] Example 2 Verification of the purification effect of TP1-FGF21 protein

[0043] (1)Screening of the high-expression system of TP1-FGF21 Escherichia coli

[0044] Pick single colonies from the transformed Escherichia coli plate and inoculate them into LB medium containing ampicillin antibiotic (ampicillin antibiotic:LB medium = 1:1000), and place them in a tabletop shaker for cultivation. When the OD 600 reaches 1.0, retain 1 ml of the bacterial liquid before induction as the pre-induction sample, then add the inducer IPTG with a final concentration of 50 mM, and continue to cultivate for 6 hours. Then, aspirate 1 ml of the bacterial liquid for centrifugation.

[0045] After centrifuging the bacterial liquid samples of Escherichia coli before and after induction, the precipitate obtained is the fusion protein. Add protein loading buffer (the volume ratio of the mixture to the protein loading buffer is 10:1), and boil in water bath for 10 min. Separate the extracted TP1-FGF21 fusion protein by SDS-PAGE gel, stain it with Coomassie Brilliant Blue staining solution, then decolorize it with decolorizing solution, and place it in an Image Lab imager for detection.

[0046] (2)Expression and purification of TP1-FGF21 fusion protein

[0047] Inoculate the transformed Escherichia coli into 50 mL of LB medium containing ampicillin antibiotic (ampicillin antibiotic:LB medium = 1:1000), and place it in a tabletop shaker at 37 °C and 200 rpm for overnight cultivation to obtain the primary seed culture solution. The next day, expand the cultivation of the primary seed solution and inoculate it into 500 mL of LB medium (ampicillin antibiotic:LB medium = 1:1000), and continue to place it in a tabletop shaker at 37 °C and 200 rpm for cultivation. When the OD 600When it reaches 1.0, 1 ml of the bacterial liquid before induction is taken as the sample before induction. Then, the inducer IPTG with a final concentration of 50 mM is added, and after continuous cultivation for 6 hours, the LB medium is collected and centrifuged at 9000 rpm and 4 °C for 30 minutes using a floor-standing centrifuge. The precipitate is the bacterial cells of the TP1-FGF21 fusion protein.

[0048] (3) The bacterial cells of the TP1-FGF21 fusion protein obtained by centrifugation are added to the cell lysis buffer (the volume ratio of the cell lysis buffer to the bacterial cells is 20:1), and broken using an ultrasonic disruptor (working for 4 s and pausing for 6 s) for 10 minutes, a total of 3 times. After breaking, it is stirred using a magnetic stirrer for 2 hours, and then centrifuged at 9000 rpm and 4 °C for 30 minutes using a floor-standing centrifuge. The collected precipitate is the cell lysis precipitate.

[0049] (4) The cell lysis precipitate is added to the cell washing buffer 1 (the volume ratio of the cell washing buffer to the bacterial cells is 20:1), and broken using an ultrasonic disruptor (working for 4 s and pausing for 6 s) for 10 minutes, a total of 3 times. After breaking, it is stirred using a magnetic stirrer for 2 hours, and then centrifuged at 9000 rpm and 4 °C for 30 minutes using a floor-standing centrifuge. The collected precipitate is the cell washing precipitate 1.

[0050] (5) The cell lysis precipitate is added to the cell washing buffer 2 (the volume ratio of the cell washing buffer to the bacterial cells is 20:1), and broken using an ultrasonic disruptor (working for 4 s and pausing for 6 s) for 10 minutes, a total of 3 times. After breaking, it is stirred using a magnetic stirrer for 2 hours, and then centrifuged at 9000 rpm and 4 °C for 30 minutes using a floor-standing centrifuge. The collected precipitate is the cell washing precipitate 2.

[0051] (6) The cell lysis precipitate is added to the cell denaturing solution (the volume ratio of the cell denaturing solution to the bacterial cells is 50:1), and broken using an ultrasonic disruptor (working for 4 s and pausing for 6 s) for 10 minutes, a total of 3 times. After breaking, it is stirred using a magnetic stirrer for 2 hours, and then centrifuged at 9000 rpm and 4 °C for 30 minutes using a floor-standing centrifuge. The collected supernatant is the TP1-FGF21 protein loading solution.

[0052] (7) After filtering the TP1-FGF21 protein loading solution through a circulating water multi-purpose vacuum pump, the protein sample solution is loaded onto an NI ion column by a protein purification system. Then, the target protein TP1-FGF21 is eluted with 50 mM and 250 mM imidazole. The eluted target protein is separated by an SDS-PAGE gel, stained with Coomassie Brilliant Blue staining solution, and then decolorized with a decolorizing solution, and detected in an Image Lab imager.

[0053] As Figure 4 shown, the purification result diagram of the TP1-FGF21 protein of the present invention. ByFigure 4 As a result, from left to right are Marker, TP1-FGF21 protein loading solution, the target protein TP1-FGF21 eluted with 50 mM imidazole, the target proteins TP1-FGF21 1 and 2 eluted with 250 mM imidazole. As can be seen from the figure, the recombinant fusion protein TP1-FGF21 can be eluted at a concentration of 250 mM imidazole, and the purified recombinant fusion protein TP1-FGF21 can be obtained.

[0054] Example 3 Verification of the Effect of UVB on Inducing DNA Damage in HaCaT Cells

[0055] (1) Establishment of the UVB-Irradiated HaCaT Cell Model

[0056] Culture HaCaT cells in a six-well plate. When the cell number reaches 80%-85%, pretreat HaCaT cells with TP1-FGF21 and FGF21 for 3 hours, and then place them in an ultraviolet irradiator for UVB (100 mJ / cm 2 ) irradiation. At the same time, set up a non-irradiated control group. Three groups of HaCaT cells are obtained, namely the control group, the comparison group, and the drug-treated group.

[0057] (2) Verification of the Effect of TP1-FGF21 on Reducing DNA Damage in HaCaT Cells

[0058] After irradiation, wash three times with PBS, place them in a cell incubator and continue to culture for 1 hour, perform immunofluorescence staining and detect the production of CPD and γ-H2AX by laser confocal microscopy.

[0059] As Figure 5 shown, the detection diagram of the effect of TP1-FGF21 of the present invention on reducing DNA damage. As Figure 5 known, compared with the non-irradiated group, UVB irradiation can cause DNA damage in HaCaT cells. After pretreatment with FGF21 and TP1-FGF21, the production of CPD and γ-H2AX can be inhibited, and compared with FGF21, TP1-FGF21 has a better effect on inhibiting the production of CPD and γ-H2AX.

[0060] Example 4 Effect of UVB on Mitochondrial Membrane Potential in Damaged HaCaT Cells

[0061] (1) Establishment of the UVB-Irradiated HaCaT Cell Model

[0062] Culture HaCaT cells in a confocal dish. When the cell number reaches 80%-85%, pretreat HaCaT cells with TP1-FGF21 and FGF21 for 3 hours, and then place them in an ultraviolet irradiator for UVB (1 J / cm 2Irradiation was performed while setting up an unirradiated control group. Three groups of HaCaT cells were obtained, namely the control group, the comparison group, and the medicated group.

[0063] (2)Verification of the effect of TP1-FGF21 on reducing ROS in HaCaT cells

[0064] After irradiation, the cells were washed three times with PBS, and then ROS staining solution (at a ratio of 1:3000) was added. 600 μL of the ROS staining solution was added to each well, and then the cells were placed back in the cell culture incubator and cultured for another 20 minutes. After that, the cells were washed three times with PBS, and then stained with Hochest staining solution for 20 minutes. After that, the cells were washed three times with PBS again, and finally observed under a confocal microscope.

[0065] As Figure 6 shown, this is the detection diagram of the effect of the TP1-FGF21 of the present invention on reducing the mitochondrial membrane potential of UVB-damaged HaCaT cells. As Figure 6 can be seen, compared with the unirradiated group, UVB irradiation can significantly increase the level of mROS in HaCaT cells. However, pretreatment with FGF21 and TP1-FGF21 can significantly inhibit the production of mROS caused by UVB irradiation, and the effect of pretreating with TP1-FGF21 to inhibit the production of mROS in HaCaT cells is better than that of pretreating with FGF21.

[0066] The above-disclosed are only the preferred embodiments of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope covered by the present invention.

Claims

1. A fibroblast growth factor 21 - penetratin fusion protein, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

1.

2. A gene encoding the fusion protein as claimed in claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A recombinant vector containing the gene as claimed in claim 2.

4. The recombinant vector according to claim 3, wherein The recombinant vector is prepared using pET15b.

5. A recombinant bacterium containing the recombinant vector as claimed in claim 3 or 4.

6. Use of the fusion protein as claimed in claim 1 in the preparation of a skin photo-damage protecting agent.

7. A preparation method for expressing the fusion protein as described in claim 1 in an E. coli system, characterized in that, The method comprises the following processes: (1) Use Nde I and Kpn I endonucleases to cut down the TP-1 gene on the vector, use Kpn I and BamH I endonucleases to cut down the FGF21 gene on the vector, use Nde I and BamH I endonucleases to cut open the pET15b vector, use a DNA recovery kit to recover the cut-down TP-1 gene, FGF21 gene and the cut-open pET15b vector, ligate with T4 ligase, transform the ligation product into Escherichia coli, screen out the cloned bacteria containing the recombinant vector as claimed in claim 3, name the recombinant vector pET15b-TP1-FGF21, and extract the plasmid of the pET15b-TP1-FGF21 vector; (2) Transfer the extracted plasmid of the pET15b-TP1-FGF21 vector into Escherichia coli competent cells by heat shock method, and use PCR method to screen out the Escherichia coli identified to contain the pET15b-TP1-FGF21 plasmid; place the identified Escherichia coli in a culture at 37°C and a rotation speed of 200 rpm until the absorbance OD600 of the Escherichia coli broth reaches 1.0, and use centrifugation method to collect 1 ml of the cultured Escherichia coli to prepare a sample before induction; (3) When OD600 reaches 1.0, add an inducer IPTG with a final concentration of 0.5 mM, continue to culture at 37°C and a rotation speed of 200 rpm, and when OD600 reaches 2.0 - 2.5, use centrifugation method to collect the cultured Escherichia coli, and take another 1 ml of the induced Escherichia coli broth to prepare an electrophoresis sample.

8. The preparation method according to claim 7, characterized in that: In step (2), screening out the Escherichia coli identified to contain the pET15b-TP1-FGF21 plasmid by PCR method comprises the following processes: Use a genomic extraction kit to extract the whole genome of the Escherichia coli into which the pET15b-TP1-FGF21 plasmid is introduced, separate the amplification product by agarose gel using upstream and downstream primers by PCR method, and detect using an ultraviolet imager; Cultivate the Escherichia coli with positive PCR identification until the OD600 reaches 1.0, then aspirate 1 ml of the bacterial liquid as the pre-induction sample, and add the inducer IPTG with a final concentration of 0.05 mM; continue to cultivate for 6 - 8 hours. When the OD600 reaches 2.0 - 2.5, aspirate 1 ml of the induced Escherichia coli bacterial liquid, centrifuge it, add the loading buffer, and perform a boiling water bath. Separate the extracted Escherichia coli protein through an SDS-PAGE gel, transfer it to a PVDF membrane using a membrane transfer instrument, add the blocking solution and block it at room temperature. Wash off the blocking solution, add the anti-FGF21 rabbit antibody, and incubate overnight at 4°C; wash off the antibody, add the alkaline phosphatase-labeled mouse antibody, and incubate at room temperature; wash off the mouse antibody, add the chromogenic agent BCIP / NBT, and place it in an imager for detection; screen out the strain of Escherichia coli transformed with pET15b-TP1-FGF21 that highly expresses the fusion protein as described in claim 1 through molecular biology detection.

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