A preparation method of amelogenin expressed in the form of inclusion bodies, and its products and applications

Abstract

The present invention provides a method for preparing amelogenin expressed in the form of inclusion bodies, a product and an application thereof, the preparation method comprising the following steps: (1) expressing a recombinant plasmid containing an amelogenin gene in an exogenous expression system to obtain a recombinant amelogenin in the form of inclusion bodies; (2) using a dissolving solution containing a concentration gradient of urea to dissolve the inclusion bodies in step (1) in a gradient manner, centrifuging to obtain a supernatant and a precipitate; (3) mixing the supernatant in step (2) with a renaturing agent, filtering, and desalting the filtrate to obtain the amelogenin expressed in the form of inclusion bodies. The present invention creatively uses a dissolving solution containing a gradient concentration of urea to separate and purify amelogenin, adds urea of ​​different concentration gradients to dissolve the inclusion bodies, and obtains a target protein with high purity and strong activity. In addition, the preparation process is simple to operate and suitable for industrial scale-up.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a preparation method, product and application of amelogenin expressed in the form of inclusion bodies. Background Art

[0002] Protein purification technology is an important technology in the field of biological research. The study of protein function is inseparable from the separation and purification of proteins. Protein purification mainly uses the similarities and differences between different proteins to remove non-protein substances and separate the target protein. At present, the more mature chromatography technologies for protein purification include affinity purification technology, ion exchange chromatography and gel filtration chromatography. In the industrial production of proteins, although these technologies are widely applicable, they often face defects such as long R&D cycle, complex procedures, high manpower and material costs, etc., which require a lot of money, manpower and time, resulting in a substantial increase in costs.

[0003] Enamel matrix proteins are a series of extracellular matrix proteins synthesized and secreted by ameloblasts differentiated from the inner enamel epithelium when the tooth develops to the bell stage. Since 1975, when it was first proposed that enamel-related proteins from the root sheath of Hertwig's epithelium could induce the formation of acellular cementum in the tooth root, in vitro experiments and clinical treatments have widely confirmed that enamel matrix proteins can effectively promote the regeneration of periodontal ligament, cementum, and alveolar bone, forming an arrangement similar to normal periodontal tissue, and achieving true periodontal regeneration. Amelogenin (Am) is the most important component of enamel matrix proteins in development and is also the main active component of enamel matrix proteins that promotes periodontal regeneration. However, the composition of Am is not single, there are at least 9 components, with a molecular weight range of 5 to 27 kDa. Compared with general recombinant proteins, recombinant amelogenin has a more stable spatial structure and physical properties.

[0004] CN101565463A discloses a recombinant human amelogenin and a preparation method thereof. The technical scheme of the invention is as follows: a recombinant human amelogenin is prepared by the following methods: a. cloning the human amelogenin mature peptide gene hAm encoded by the X chromosome; b. constructing a recombinant prokaryotic expression plasmid PGEX4T1-hAm containing the above gene; c. transforming the recombinant prokaryotic expression plasmid PGEX4T1-hAm obtained in step b into an expression Escherichia coli strain; d. inducing the cloned expression strain obtained in step c to express the fusion protein GST-hAm; e. purifying the fusion protein by GST affinity chromatography. The invention cloned the coding sequence of the mature peptide of human amelogenin from human deciduous tooth germ tissue for the first time, and successfully expressed the fusion protein in Escherichia coli by the prokaryotic expression plasmid. The artificially synthesized recombinant human amelogenin can be obtained by GST affinity chromatography purification, but it is necessary to further remove the GST protein tag by enzyme cutting and purification steps.

[0005] CN103014050A discloses a recombinant porcine amelogenin, which is encoded by a full-length porcine amelogenin gene, and the preparation method is as follows: synthesizing the full-length porcine amelogenin gene; constructing a recombinant prokaryotic expression plasmid PGEX4T1-pAm containing the above gene; transforming the obtained recombinant prokaryotic expression plasmid PGEX4T1-pAm into an Escherichia coli strain; inducing the obtained recombinant strain to express the fusion protein GST-pAm; removing the GST tag protein of the obtained fusion protein GST-pAm, and obtaining the recombinant porcine amelogenin pAm. The coding sequence of the full-length porcine amelogenin was synthesized for the first time, and the fusion protein was successfully expressed in Escherichia coli through a prokaryotic expression plasmid. The cloned expression strain obtained by this invention can be preserved for a long time and replicated in large quantities at any time. It can conveniently and quickly synthesize a large amount of recombinant porcine amelogenin artificially, and also provides the possibility for mass production and commercialization of amelogenin in the future. However, this scheme is similar to the amelogenin purification scheme disclosed in CN101565463A. Both require enzymatic cleavage and purification steps to remove the GST protein tag and expose the amelogenin to realize the complete biological function.

[0006] At present, the purification of amelogenin faces problems such as long R&D cycle, complex procedures, and high manpower and material costs. Therefore, it is very meaningful to utilize the characteristics of amelogenin to develop a simple and efficient amelogenin purification method. Summary of the invention

[0007] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for preparing amelogenin expressed in the form of inclusion bodies, as well as a product and application thereof.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for preparing amelogenin expressed in the form of inclusion bodies, the preparation method comprising the following steps:

[0010] (1) expressing a recombinant plasmid containing an amelogenin gene in an exogenous expression system to obtain a recombinant amelogenin in the form of inclusion bodies;

[0011] (2) using a dissolving solution containing urea in a gradient concentration to dissolve the inclusion bodies in step (1) in a gradient manner, and centrifuging to obtain a supernatant and a precipitate;

[0012] (3) mixing the supernatant in step (2) with a renaturing agent, filtering, and desalting the filtrate to obtain the amelogenin expressed in the form of inclusion bodies.

[0013] The present invention creatively uses a dissolving solution containing gradient concentrations of urea to separate and purify amelogenin, wherein urea is a relatively strong denaturant, which can break various chemical bonds within and between inclusion body protein molecules through ionic interactions, stretching the polypeptide, and turning the inclusion body into a soluble protein. Urea with different concentration gradients is added to dissolve the inclusion body, and the obtained target protein has high purity and strong activity. In addition, the preparation process is simple to operate and suitable for industrial scale-up.

[0014] Preferably, the exogenous expression system in step (1) comprises a prokaryotic expression system.

[0015] Preferably, the prokaryotic expression system comprises Escherichia coli, Bacillus subtilis or Lactobacillus.

[0016] Preferably, the amelogenin gene is an amelogenin gene of human, bovine, porcine or dog origin.

[0017] Preferably, the components of the dissolving solution in step (2) further include any one of Tris, TritonX-100, β-mercaptoethanol or EDTA-2Na, or a combination of at least two of them. The combination of at least two includes a combination of Tris and TritonX-100, a combination of TritonX-100 and β-mercaptoethanol, or a combination of β-mercaptoethanol and EDTA-2Na, and any other combinations may be selected and will not be described in detail here.

[0018] The components of the dissolving solution in step (2) of the present invention also include any one or a combination of at least two of Tris, TritonX-100, β-mercaptoethanol or EDTA-2Na, wherein Tris is an alkaline buffer salt, which can help maintain the pH of the solution within a certain range and help stabilize the protein structure; TritonX-100 is a mild surfactant that can dissolve proteins on the cell membrane, wash away cell lipids and membrane proteins in the inclusion body, and play a role in removing impurities; β-mercaptoethanol is a strong reducing agent that can open the mismatched disulfide bonds in the inclusion body, help the folding of the correct structure during the inclusion body renaturation process, and reduce the protein precipitation caused by protein misfolding during the renaturation process; EDTA-2Na is a metal chelating agent that can chelate metal ions in the solution, effectively prevent oxidation caused by metals, and is an antioxidant synergist. It can also inhibit the activity of proteases and the growth of microorganisms, and effectively alleviate protein degradation. TritonX-100 can effectively dissolve inclusion body proteins and wash away impurities in urea with a concentration gradient, and the obtained amelogenin has high purity. β-Mercaptoethanol and EDTA-2Na complement each other and can enhance their effects synergistically. Through the synergistic effect between the components in preventing protein oxidation, the purified amelogenin has strong activity. Furthermore, the more active amelogenin has stronger anti-inflammatory and repair effects.

[0019] Preferably, the mass volume ratio of the inclusion bodies in step (2) to the dissolving solution used for a single dissolution is 1:(8-15) g / mL.

[0020] The specific numerical value in (8-15) can be selected as 8, 9, 10, 11, 12, 13, 14 or 15, etc. Other specific point values ​​within the above numerical ranges can be selected, and they will not be described one by one here.

[0021] Preferably, the gradient range of urea concentration in the dissolving solution in step (2) is 2-6M.

[0022] The specific values ​​of 2-6M can be selected as 2M, 3M, 4M, 5M or 6M, etc. Other specific point values ​​within the above numerical ranges can be selected, and they will not be described one by one here.

[0023] The amelogenin prepared when the urea concentration gradient of the present invention is 2-6M has higher protein purity and stronger protein activity, and further has stronger anti-inflammatory and repair effects.

[0024] Preferably, the sequential gradient dissolution in step (2) is a sequential gradient dissolution of urea concentration from low to high.

[0025] When the sequential gradient dissolution described in the present invention is a gradient dissolution from low to high urea concentration, the target protein can be fully separated and purified, thereby obtaining a target protein with higher purity.

[0026] Preferably, the initial concentration of urea in step (2) is 2M.

[0027] In the present invention, the initial concentration of urea is 2M. When the initial concentration of urea is selected to be below 2M, the solubility of the target protein is low, and the purity of the amelogenin obtained by separation and purification is low.

[0028] Preferably, the dissolving in step (2) is accompanied by shaking.

[0029] Preferably, the shaking time is 1.5-3h.

[0030] The specific numerical value of 1.5-3h can be selected as 1.5h, 1.7h, 1.9h, 2.1h, 2.3h, 2.5h, 2.7h, 2.9h or 3h, etc. Other specific point values ​​within the above numerical ranges can be selected, and they will not be described one by one here.

[0031] Preferably, step (2) further includes a washing operation before the dissolving.

[0032] Preferably, the washing solution used for the washing comprises Tris and / or TritonX-100.

[0033] In the separation and purification process of the present invention, a washing solution containing Tris and / or TritonX-100 is used to wash the precipitate before dissolution, which can effectively remove impurities such as culture medium and foreign proteins, and the obtained target protein has higher purity.

[0034] Preferably, the pH of the washing liquid is 7-10.

[0035] The specific values ​​in 7-10 can be selected from 7, 7.3, 7.6, 7.9, 8.2, 8.5, 8.8, 9.1, 9.4, 9.7 or 10, etc. Other specific point values ​​within the above numerical ranges can be selected, and they will not be described one by one here.

[0036] Preferably, the gradient dissolution in step (2) is performed 2-5 times.

[0037] The specific value among the 2-5 times can be selected as 2 times, 3 times, 4 times or 5 times.

[0038] Preferably, the centrifugal speed in step (2) is 8000-15000 rpm, and the centrifugal time is 15-45 min.

[0039] The specific value of 8000-15000rpm can be 8000rpm, 9000rpm, 10000rpm, 12000rpm, 13000rpm, 14000rpm or 15000rpm, etc. Other specific point values ​​within the above numerical ranges can be selected and will not be described one by one here.

[0040] The specific value of 15-45min can be selected as 15min, 20min, 25min, 30min, 35min, 40min or 45min, etc. Other specific point values ​​within the above numerical ranges can be selected, and they will not be described one by one here.

[0041] Preferably, the specific operation of the mixing in step (3) is: dropping the supernatant into the refolding agent.

[0042] Preferably, the renaturing agent in step (3) includes Tris, NaCl, glycerol and EDTA-2Na, and also includes L-cysteine.

[0043] The refolding agent in step (3) of the present invention includes Tris, NaCl, glycerol and EDTA-2Na, and also includes L-cysteine, wherein Tris is an alkaline pH buffer solution, which can prevent the protonation of free thiols from affecting the formation of correctly paired disulfide bonds. At the same time, the alkaline condition is far away from the isoelectric point of amelogenin, which can enhance the solubility of amelogenin; NaCl is a common neutral salt, and a certain concentration of salt can increase the surface charge of protein molecules, enhance the interaction between protein molecules and water molecules, form a hydration film around the protein, enhance the stability of the protein, and increase the solubility of the protein in the aqueous solution; glycerol is a common organic solvent, which can stabilize the structure of the protein through hydration, and glycerol can increase the viscosity of the refolding solution and reduce the mutual collision between proteins. , thereby preventing excessive aggregation of proteins to form precipitation and improving the renaturation efficiency of inclusion bodies; L-cysteine ​​is a smaller redox partner that can promote the rapid exchange reaction of incorrectly formed disulfide bonds, thereby increasing the yield of correctly paired disulfide bonds, which can not only regulate the balance of protein folding and aggregation processes in cells, but also promote protein folding and renaturation in vitro, thereby improving the yield in the inclusion body renaturation process; EDTA-2Na is a metal chelator, which, in addition to its antioxidant effect, can also inhibit the activity of proteases and the growth of microorganisms, and effectively alleviate protein degradation; the present invention creatively finds that NaCl, glycerol, EDTA-2Na and L-cysteine ​​complement each other and synergistically enhance each other, and the renaturation agent containing four ingredients has a better effect in improving protein purity and enhancing protein activity.

[0044] Preferably, the pH of the renaturing agent in step (3) is 7-9.

[0045] The specific values ​​among 7-9 can be selected as 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8 or 9, etc. Other specific point values ​​within the above numerical ranges can be selected, and they will not be described one by one here.

[0046] Preferably, the step of dropping the supernatant into the refolding agent is accompanied by stirring.

[0047] Preferably, the pore size of the filter membrane used in the filtration in step (3) is 0.45 μm.

[0048] Preferably, the desalting column used in step (3) is a G25 desalting column.

[0049] In a second aspect, the present invention provides an amelogenin product expressed in the form of inclusion bodies, wherein the amelogenin product comprises the amelogenin prepared by the preparation method described in the first aspect.

[0050] Preferably, the amelogenin product further comprises a lyoprotectant.

[0051] Preferably, the lyoprotectant includes any one of mannitol, trehalose, sucrose or EDTA-2Na, or a combination of at least two of them. The combination of at least two includes a combination of mannitol and trehalose, a combination of trehalose and sucrose, or a combination of sucrose and EDTA-2Na, and any other combinations may be selected and will not be described in detail here.

[0052] In a third aspect, the present invention provides a preparation method as described in the first aspect or a use of the amelogenin product as described in the second aspect in the preparation of oral care, oral medical or beauty skin care products.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] The present invention creatively uses a dissolving solution containing gradient concentrations of urea to separate and purify amelogenin, wherein urea is a relatively strong denaturant, which can break various chemical bonds within and between inclusion body protein molecules through ionic interactions, stretching the polypeptide, and turning the inclusion body into a soluble protein. Urea with different concentration gradients is added to dissolve the inclusion body, and the obtained target protein has high purity and strong activity. In addition, the preparation process is simple to operate and suitable for industrial scale-up. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The left picture is the product without adding lyophilizing protective agent in Example 1, and the right picture is the product with adding lyophilizing protective agent in Example 1.

[0056] Figure 2 This is the SDS-PAGE gel electrophoresis protein gel image of amelogenin expressed in inclusion body form obtained in Example 1.

[0057] Figure 3 These are the zebrafish anti-inflammatory results corresponding to the blank control group (a), positive control group (b) and Example 1 group (c) in Test Example 2.

[0058] Figure 4 The zebrafish tail repair results corresponding to the blank control group (a), the positive control group (b) and the Example 1 group (c) in Test Example 3 are shown. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0060] The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention has no particular restrictions. The experimental methods without specifying specific conditions in the embodiments are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0061] Unless otherwise specified, all technical terms and scientific terms used in this specification have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. However, in case of any conflict, the present specification including the definitions shall prevail.

[0062] The Escherichia coli (BL21 (DE3)) in the following preparation examples, embodiments, and test examples was purchased from Suzhou Hongxun Biotechnology Co., Ltd., the defoaming agent was purchased from Guangzhou Ye Zhixin Biotechnology Co., Ltd., and tricarboxylic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0063] Preparation Example 1

[0064] Preparation of recombinant human amelogenin fermentation strain

[0065] The amino acid sequence of human amelogenin was searched from the NCBI database as shown in SEQ ID NO: 1. The expression vector selected was the pET-28a vector suitable for the Escherichia coli host and sent to a sequencing company for synthesis to obtain a recombinant human amelogenin expression vector.

[0066] The amino acid sequence shown in SEQ ID NO: 1 is:

[0067] MGTWILFACLLGAAFAMPLPPHPGHPGYINFSYEVLTPLKWYQSIRPPYPSYGYEPMGGWLHHQIIPVLSQQHPPTHTLQPHHHIPVVPAQQPVI PQQPMMPVPGQHSMTPIQHHQPNLPPPAQQPYQPQPVQPQPHQPMQPQPPVHPMQPLPPQPPLPPMFPMQPLPPMLPDLTLEAWPSTDKTKREEVD

[0068] The above expression vector was transformed into Escherichia coli (BL21 (DE3)) strain by chemical transformation, and the positive clones in the transformed plate colonies were screened by SDS-PAGE gel electrophoresis. The screened positive clones were transferred to 2 mL LB medium (50 mg / mL kanamycin was added at a volume ratio of 1:1000), and cultured at 37°C, 220 rpm for 12 h. The cultured bacterial solution was diluted 1 times with 50% glycerol by volume, dispensed into freezing tubes, and frozen at -80°C to obtain a recombinant human amelogenin fermentation strain.

[0069] Preparation Example 2

[0070] Fermentation process of recombinant human amelogenin fermentation strain

[0071] The recombinant human amelogenin fermentation strain was inoculated into 50 ml LB medium (50 mg / mL kanamycin was added at a volume ratio of 1:1000) and cultured at 37°C and 220 rpm for 4-6 h until the bacterial solution OD 600 Reach 0.8 and prepare seed solution.

[0072] The seed solution was inoculated into 1000 ml LB medium (50 mg / mL kanamycin was added at a volume ratio of 1:1000) at a volume ratio of 1:1000, and cultured at 37° C. and 220 rpm for 12 h to prepare a secondary seed solution.

[0073] The secondary seed solution was inoculated into a 50L fermenter at a volume ratio of 1:10. The fermenter contained 20L of fermenter medium (50mg / mL kanamycin was added to the medium at a volume ratio of 1:1000). The pH was adjusted to 7 with NaOH. The speed of the fermenter was controlled at 500rpm and the temperature was controlled at 37°C. The OD of the fermentation liquid in the tank was monitored every hour after loading. 600 Value and pH value, when the fermentation broth OD 600 When the temperature reached 8, 1 M IPTG inducer was added at a volume ratio of 1:1000, and the culture was continued for 6 h. Then the fermentation was terminated and the fermentation broth was collected.

[0074] The fermentation broth was centrifuged at 15,000 rpm using a centrifuge, the supernatant was discarded, the fermentation cells were weighed, and the fermentation cells were frozen in a -80°C refrigerator.

[0075] The fermentation tank culture medium formula is as follows: tryptone 23g / L, yeast powder 23g / L, glucose 2g / L, sodium chloride 5g / L, magnesium sulfate 1g / L, calcium chloride 0.02g / L, ammonium chloride 0.5g / L, sodium dihydrogen phosphate 10mmol / L, defoaming agent 1%.

[0076] Example 1

[0077] This embodiment provides a kind of amelogenin product, and its preparation method comprises the following steps:

[0078] (1) Use 10 parts of 20 mM Tris buffer with a pH of 8.5 to resuspend 1 part of the fermentation bacterial precipitate prepared in Preparation Example 2, and use a high-pressure homogenizer at 800 bar to break the bacterial cells in the resuspended liquid three times until the bacterial liquid is clear and transparent, and observe under a microscope to ensure that the bacterial cells are broken. Then, the bacterial liquid is centrifuged at 14000 rpm for 60 min to collect the solid precipitate.

[0079] (2) Add washing solution at a ratio of solid precipitate: washing solution (20 mM Tris buffer, pH 9.0 + 0.1% Triton X-100) = 1:50 (g / mL), shake for 2 hours, centrifuge at 8000 rpm and 20°C for 30 minutes, collect and weigh the shaken solid precipitate. Repeat this step once.

[0080] (3) Add dissolving solution A (20 mM Tris buffer with a pH of 9.0 + 2 M urea + 1% TritonX-100 + 1 mM β-mercaptoethanol + 0.1 mM EDTA-2Na) at a ratio of precipitate to dissolving solution A = 1:10 (g / mL) in step (2), shake for 2 h, and centrifuge at 14000 rpm and 20°C for 30 min to collect supernatant A and solid precipitate A.

[0081] According to the ratio of solid precipitate A: dissolving solution B = 1:10 (g / mL), dissolving solution B (20mM Tris buffer with pH 9.0 + 4M urea + 1% TritonX-100 + 1mM β-mercaptoethanol + 0.1mM EDTA-2Na) was added, and the mixture was shaken for 2h. Centrifuged at 14000rpm and 20℃ for 30min, and the supernatant B and solid precipitate B were collected.

[0082] Dissolving solution C (20 mM Tris buffer with a pH of 9.0 + 6 M urea + 1% TritonX-100 + 1 mM β-mercaptoethanol + 0.1 mM EDTA-2Na) was added at a ratio of solid precipitate B: dissolving solution C = 1:10 (g / mL), shaken for 2 h, centrifuged at 14000 rpm and 20 ° C for 30 min, and the supernatant C and solid precipitate C were collected.

[0083] The supernatants A, B and C were mixed and slowly dripped into the renaturation agent (the renaturation agent contained 20mM Tris buffer, 200mM NaCl, 1mM L-cysteine, 0.1mM EDTA-2Na and 10% glycerol by volume, and the pH of the renaturation agent was adjusted to 8.0) frozen to 4°C at a volume ratio of 1:100, and the mixture was dripped while stirring until the liquid was uniform and there was no obvious change. Subsequently, the filtrate was filtered using a 0.45μm filter membrane, and the filtrate was desalted using a G25 gel desalting column to obtain amelogenin expressed in the form of inclusion bodies.

[0084] (4) Mix 48 parts of mannitol, 12 parts of trehalose, 12 parts of sucrose, and 0.02 parts of EDTA-2Na to prepare a lyophilization protective agent.

[0085] (5) Mix one portion of the amelogenin expressed in the form of inclusion bodies obtained in step (3) and one portion of the lyophilization protective agent obtained in step (4), and place the mixture in a freeze dryer for vacuum freeze drying to obtain an amelogenin product (the product appearance is shown in FIG. Figure 1 ).

[0086] Example 2

[0087] This embodiment provides a kind of amelogenin product, and its preparation method comprises the following steps:

[0088] (1) Use 10 parts of 20 mM Tris buffer with a pH of 8.5 to resuspend one part of the fermentation bacterial precipitate prepared in Preparation Example 2, and use a high-pressure homogenizer at 800 bar to break the bacterial cells in the resuspended liquid three times until the bacterial liquid is clear and transparent, and observe under a microscope to ensure that the bacterial cells are broken. Then, the bacterial liquid is centrifuged at 12000 rpm for 60 min to collect the solid precipitate.

[0089] (2) Add washing solution at a ratio of solid precipitate: washing solution (20 mM Tris buffer, pH 10.0 + 0.1% Triton X-100) = 1:60 (g / mL), shake for 3 hours, centrifuge at 8000 rpm and 20°C for 25 minutes, collect and weigh the solid precipitate after shaking. Repeat this step once.

[0090] (3) Add dissolving solution A (20 mM Tris buffer with a pH of 9.0 + 2 M urea + 1% TritonX-100 + 1 mM β-mercaptoethanol + 0.1 mM EDTA-2Na) at a ratio of precipitate to dissolving solution A = 1:8 (g / mL) in step (2). After shaking for 3 h, centrifuge at 8000 rpm and 20°C for 45 min to collect supernatant A and solid precipitate A.

[0091] According to the ratio of solid precipitate A: dissolving solution B = 1:8 (g / mL), dissolving solution B (20mM Tris buffer with pH 9.0 + 4M urea + 1% TritonX-100 + 1mM β-mercaptoethanol + 0.1mM EDTA-2Na) was added, and the mixture was shaken for 1.5h. Centrifuged at 8000rpm and 20℃ for 45min, and the supernatant B and solid precipitate B were collected.

[0092] The supernatants A and B were mixed and slowly dripped into the renaturation agent (the renaturation agent contained 20mM Tris buffer, 200mM NaCl, 1mM L-cysteine, 0.1mM EDTA-2Na and 10% glycerol by volume, and the pH of the renaturation agent was adjusted to 9.0) frozen to 4°C at a volume ratio of 1:100, and the mixture was dripped while stirring until the liquid was uniform and there was no obvious change. Subsequently, the mixture was filtered using a 0.45μm filter membrane, and the filtrate was desalted using a G25 gel desalting column to obtain amelogenin expressed in the form of inclusion bodies.

[0093] (4) Mix 40 parts of mannitol, 15 parts of trehalose, 5 parts of sucrose, and 0.02 parts of EDTA-2Na to prepare a lyophilization protective agent.

[0094] (5) Mix one portion of the amelogenin expressed in the form of inclusion bodies obtained in step (3) and one portion of the lyophilization protective agent obtained in step (4), and place the mixture in a freeze dryer for vacuum freeze drying to obtain an amelogenin product.

[0095] Example 3

[0096] This embodiment provides a kind of amelogenin product, and its preparation method comprises the following steps:

[0097] (1) Use 10 parts of 20 mM Tris buffer with a pH of 8.5 to resuspend 1 part of the fermentation bacterial precipitate prepared in Preparation Example 2, and use a high-pressure homogenizer at 800 bar to break the bacterial cells in the resuspended liquid three times until the bacterial liquid is clear and transparent, and observe under a microscope to ensure that the bacterial cells are broken. Then, the bacterial liquid is centrifuged at 13000 rpm for 50 min to collect the solid precipitate.

[0098] (2) Add washing solution at a ratio of solid precipitate: washing solution (20 mM Tris buffer, pH 7.0 + 0.1% Triton X-100) = 1:40 (g / mL), shake for 2.5 hours, centrifuge at 8000 rpm and 20°C for 35 minutes, collect and weigh the shaken solid precipitate. Repeat this step once.

[0099] (3) Add dissolving solution A (20 mM Tris buffer with a pH of 9.0 + 2 M urea + 1% TritonX-100 + 1 mM β-mercaptoethanol + 0.1 mM EDTA-2Na) at a ratio of precipitate to dissolving solution A = 1:15 (g / mL) in step (2), shake for 1.5 h, and centrifuge at 15000 rpm and 20°C for 15 min to collect supernatant A and solid precipitate A.

[0100] According to the ratio of solid precipitate A: dissolving solution B = 1:15 (g / mL), dissolving solution B (20mM Tris buffer with pH 9.0 + 3M urea + 1% TritonX-100 + 1mM β-mercaptoethanol + 0.1mM EDTA-2Na) was added, and the mixture was shaken for 3h. Centrifuged at 15000rpm and 20℃ for 15min, and the supernatant B and solid precipitate B were collected.

[0101] Dissolving solution C (20 mM Tris buffer with a pH of 9.0 + 4 M urea + 1% TritonX-100 + 1 mM β-mercaptoethanol + 0.1 mM EDTA-2Na) was added at a ratio of solid precipitate B: dissolving solution C = 1:15 (g / mL), shaken for 1.5 h, centrifuged at 15000 rpm and 20 ° C for 15 min, and the supernatant C and solid precipitate C were collected.

[0102] Dissolving solution D (20 mM Tris buffer with a pH of 9.0 + 5 M urea + 1% TritonX-100 + 1 mM β-mercaptoethanol + 0.1 mM EDTA-2Na) was added at a ratio of solid precipitate C: dissolving solution D = 1:15 (g / mL), shaken for 3 h, centrifuged at 15000 rpm and 20°C for 15 min, and the supernatant D and solid precipitate D were collected.

[0103] Dissolving solution E (20 mM Tris buffer with a pH of 9.0 + 6 M urea + 1% TritonX-100 + 1 mM β-mercaptoethanol + 0.1 mM EDTA-2Na) was added at a ratio of solid precipitate D: dissolving solution E = 1:15 (g / mL), shaken for 3 h, centrifuged at 15000 rpm and 20°C for 15 min, and the supernatant E and solid precipitate E were collected.

[0104] The supernatants A, B, C, D and E were mixed and slowly dripped into the renaturation agent (the renaturation agent contained 20mM Tris buffer, 200mM NaCl, 1mM L-cysteine, 0.1mM EDTA-2Na and 10% glycerol by volume, and the pH of the renaturation agent was adjusted to 7.0) frozen to 4°C at a volume ratio of 1:100, and the mixture was dripped while stirring until the liquid was uniform and there was no obvious change. Subsequently, the filtrate was filtered using a 0.45μm filter membrane, and the filtrate was desalted using a G25 gel desalting column to obtain amelogenin expressed in the form of inclusion bodies.

[0105] (4) Mix 50 parts of mannitol, 5 parts of trehalose, 12 parts of sucrose, and 0.01 parts of EDTA-2Na to prepare a lyophilization protective agent.

[0106] (5) Mix one portion of the amelogenin expressed in the form of inclusion bodies obtained in step (3) and one portion of the lyophilization protective agent obtained in step (4), and place the mixture in a freeze dryer for vacuum freeze drying to obtain an amelogenin product.

[0107] Example 4

[0108] The present embodiment provides amelogenin product, and the preparation method thereof is different from that of the embodiment 1 only in that after collecting the supernatant C and the solid precipitate C in step (3), a dissolution process is added: the dissolution solution D (20 mM Tris buffer with a pH of 9.0 + 8 M urea + 1% TritonX-100 + 1 mM β-mercaptoethanol + 0.1 mM EDTA-2Na) is added at a ratio of solid precipitate C: dissolution solution D = 1:10 (g / mL), shaken for 2 hours, centrifuged at 14000 rpm and 20°C for 30 minutes, the supernatant D and the solid precipitate D are collected, the supernatants A, B, C and D are mixed, and the refolding agent is added dropwise, and the rest is the same as the embodiment 1.

[0109] Example 5

[0110] This embodiment provides an amelogenin product, and the preparation method thereof is different from that of Embodiment 1 only in that 2M urea in step (3) is replaced by 1M urea, and the rest is the same as that of Embodiment 1.

[0111] Example 6

[0112] This embodiment provides an amelogenin product, and the preparation method thereof is different from that of Embodiment 1 only in that the dissolving solution does not contain Triton X-100, and the mass fraction of Triton X-100 is distributed to urea, and the rest is the same as that of Embodiment 1.

[0113] Example 7

[0114] This embodiment provides an amelogenin product, and the preparation method thereof is different from that of Embodiment 1 only in that the dissolving solution does not contain β-mercaptoethanol, and the mass fraction of β-mercaptoethanol is allocated to EDTA-2Na, and the rest is the same as that of Embodiment 1.

[0115] Example 8

[0116] This embodiment provides an amelogenin product, and the preparation method thereof is different from that of Embodiment 1 only in that the dissolving solution does not contain EDTA-2Na, and the mass fraction of EDTA-2Na is allocated to β-mercaptoethanol, and the rest is the same as that of Embodiment 1.

[0117] Example 9

[0118] This embodiment provides an amelogenin product, and its preparation method is different from that of Example 1 only in that the refolding agent component does not contain L-cysteine, and the mass fraction of L-cysteine ​​is proportionally distributed to NaCl, glycerol, and EDTA-2Na, and the rest is the same as Example 1.

[0119] Example 10

[0120] This embodiment provides an amelogenin product, and its preparation method is different from that of Example 1 only in that the refolding agent component does not contain NaCl, glycerol, and EDTA-2Na, and the mass fractions of NaCl, glycerol, and EDTA-2Na are all allocated to L-cysteine, and the rest are the same as in Example 1.

[0121] Comparative Example 1

[0122] This comparative example provides an amelogenin product, and the preparation method thereof is different from that of Example 1 only in that urea is not included in the components of the dissolving solution, and the mass fraction of urea is allocated to TritonX-100, and the rest is the same as that of Example 1.

[0123] Comparative Example 2

[0124] This comparative example provides a kind of amelogenin product, and its preparation method is different from that of Example 1 only in that the dissolution process of the dissolving solution does not use gradient urea solution treatment, but only uses a single urea solution with a concentration of 2M for treatment, that is, the specific operation steps in step (3) in Example 1 are replaced by: according to the ratio of precipitate: dissolving solution A = 1:10 (g / mL) in step (2), dissolving solution A (20mM Tris buffer with a pH of 9.0 + 2M urea + 1% TritonX-100 + 1mM β-mercaptoethanol + 0.1mM EDTA-2Na) is added, after shaking for 2h, centrifuged at 14000rpm and 20℃ for 30min, and the supernatant A and solid precipitate A are collected.

[0125] The supernatant A was slowly dripped into the renaturation agent (the renaturation agent contained 20mMTris buffer, 200mM NaCl, 1mM L-cysteine, 0.1mM EDTA-2Na and 10% glycerol by volume, and the pH of the renaturation agent was adjusted to 8.0) frozen to 4°C at a volume ratio of 1:100, and the mixture was dripped while stirring until the liquid was uniform and there was no obvious change. Subsequently, the supernatant A was filtered using a 0.45μm filter membrane, and the filtrate was desalted using a G25 gel desalting column to obtain amelogenin expressed in the form of inclusion bodies. The rest was the same as in Example 1.

[0126] Comparative Example 3

[0127] This comparative example provides amelogenin product, and its preparation method is different from that of Example 1 only in that the dissolution process of the dissolving solution does not use a gradient urea solution for treatment, but only uses a single urea solution with a concentration of 4M for treatment, that is, the specific operation steps in step (3) in Example 1 are replaced by: according to the ratio of precipitate: dissolving solution A = 1:10 (g / mL) in step (2), dissolving solution A (20mM Tris buffer with a pH of 9.0 + 4M urea + 1% TritonX-100 + 1mM β-mercaptoethanol + 0.1mM EDTA-2Na) is added, after shaking for 2h, centrifuged at 14000rpm and 20℃ for 30min, and the supernatant A and solid precipitate A are collected.

[0128] The supernatant A was slowly dripped into the renaturation agent (the renaturation agent contained 20mMTris buffer, 200mM NaCl, 1mM L-cysteine, 0.1mM EDTA-2Na and 10% glycerol by volume, and the pH of the renaturation agent was adjusted to 8.0) frozen to 4°C at a volume ratio of 1:100, and the mixture was dripped while stirring until the liquid was uniform and there was no obvious change. Subsequently, the supernatant A was filtered using a 0.45μm filter membrane, and the filtrate was desalted using a G25 gel desalting column to obtain amelogenin expressed in the form of inclusion bodies. The rest was the same as in Example 1.

[0129] Comparative Example 4

[0130] This comparative example provides amelogenin product, and its preparation method is different from that of Example 1 only in that the dissolution process of the dissolving solution does not use a gradient urea solution for treatment, but only uses a single urea solution with a concentration of 6M for treatment, that is, the specific operation steps in step (3) in Example 1 are replaced by: according to the ratio of precipitate: dissolving solution A = 1:10 (g / mL) in step (2), dissolving solution A (20mM Tris buffer with a pH of 9.0 + 6M urea + 1% TritonX-100 + 1mM β-mercaptoethanol + 0.1mM EDTA-2Na) is added, after shaking for 2h, centrifuged at 14000rpm and 20℃ for 30min, and the supernatant A and solid precipitate A are collected.

[0131] The supernatant A was slowly dripped into the renaturation agent (the renaturation agent contained 20mMTris buffer, 200mM NaCl, 1mM L-cysteine, 0.1mM EDTA-2Na and 10% glycerol by volume, and the pH of the renaturation agent was adjusted to 8.0) frozen to 4°C at a volume ratio of 1:100, and the mixture was dripped while stirring until the liquid was uniform and there was no obvious change. Subsequently, the supernatant A was filtered using a 0.45μm filter membrane, and the filtrate was desalted using a G25 gel desalting column to obtain amelogenin expressed in the form of inclusion bodies. The rest was the same as in Example 1.

[0132] Test Example 1

[0133] The purity of the amelogenin expressed in the form of inclusion bodies obtained in each of Examples 1-10 or Comparative Examples 1-4 was evaluated. The amelogenin expressed in the form of inclusion bodies obtained in step (3) of each of the Examples and Comparative Examples was taken and protein separation was performed using SDS-PAGE electrophoresis. The protein gel was then scanned using a grayscale scanning method. The proportion of amelogenin in the entire lane was the purity of the amelogenin. Figure 2 This is the SDS-PAGE electrophoresis protein gel image of amelogenin obtained in Example 1, which includes two lanes. The left side is the sample lane of Example 1, and the right side is the protein Maker lane. The order of the Maker lane from top to bottom is 150kDa, 100kDa, 70kDa, 50kDa, 40kDa, 35kDa, 25kDa, 20kDa, and 15kDa.

[0134] Table 1

[0135] Group Protein purity (%) Example 1 92.32 Example 2 90.46 Example 3 87.92 Example 4 70.35 Example 5 72.52 Example 6 67.45 Example 7 80.35 Example 8 76.58 Example 9 74.87 Example 10 72.59 Comparative Example 1 4.65 Comparative Example 2 57.85 Comparative Example 3 59.54 Comparative Example 4 62.56

[0136] As can be seen from the data in Table 1, the urea concentration of the dissolving solution used in Example 4 when purifying amelogenin exceeds 6M, and the purity of its amelogenin is lower than that of Example 1, indicating that too high a urea concentration will cause the purified product to contain more impurities; the dissolving solutions of Example 6 and Comparative Example 1 do not contain TritonX-100 and urea, respectively, and the purity of its amelogenin is lower than that of Example 1, indicating that TritonX-100 and urea can synergistically enhance the effect, fully dissolving amelogenin while ensuring the purity of amelogenin. The refolding agent of Example 9 does not contain L-cysteine, and the refolding agent of Example 10 does not contain NaCl, glycerol, and EDTA-2Na, and the purity of its amelogenin is lower than that of Example 1, indicating that L-cysteine ​​can cooperate with NaCl, glycerol, and EDTA-2Na to have a synergistic effect in improving the purity of the purified amelogenin product. Compared with Example 1, the initial urea concentration of Example 5 is 1 M, which is lower than 2 M of Example 1, and the protein purity is lower than that of Example 1, indicating that when the initial urea concentration is 2 M, it is beneficial to improve the purity of amelogenin. Comparative Examples 2-4 use a single concentration of urea dissolving solution, and do not perform concentration gradient dissolution. The protein purity is lower than that of Example 1, indicating that the purity of amelogenin prepared by the present invention using a dissolving solution with a urea concentration gradient is higher.

[0137] Test Example 2

[0138] The anti-inflammatory effect of the amelogenin expressed in the form of inclusion bodies obtained in the examples and comparative examples was evaluated by the following specific method:

[0139] (1) Select zebrafish that are 3 days after fertilization and have normal development, and randomly distribute them into 96-well plates, 5 per well, and 3 replicates per group. Divide into blank control group, inflammatory model group, experimental group and positive control group. Add 200 μL of zebrafish culture water (5mM NaCl, 0.17mM KCl, 0.33mM CaCl2, 0.33mM MgSO4·7H2O) to each well of the blank control group and the inflammatory model group, add 200 μL of 0.03g / L dexamethasone solution to each well of the positive control group, and add 200uL of 0.03g / L test solution prepared in each embodiment and comparative example to each well of the remaining groups, cover the culture plate panel, and incubate in a 28.5℃ biochemical incubator for 60min. Both the test substance and the positive control are prepared with zebrafish culture water.

[0140] (2) Remove the solution in the 96-well plate, add 200 μL of zebrafish culture water to the blank control group, add 200 μL of 20 μM copper sulfate solution to the inflammation model group, add 200 μL of a mixed solution of copper sulfate and dexamethasone to the positive control group, add 200 μL of a mixed solution of copper sulfate and the test substance solution to the experimental group, cover the culture panel, and incubate in a 28.5°C biochemical incubator for 15 minutes. The copper sulfate concentration in the above mixed solution is 20 μM, the test substance and dexamethasone concentrations are both 0.03 g / L, and the solvent is zebrafish culture water.

[0141] (3) After incubation, zebrafish in each group were anesthetized with 0.0002 g / mL tricaine, placed on a glass slide, fixed in a lateral position, and observed and photographed under a fluorescence microscope.

[0142] (4) After taking the photos, the image processing software Image Pro Plus was used to count the number of neutrophils in each group of zebrafish that migrated to the lateral line and calculate the inflammatory cell inhibition rate.

[0143] The use of copper sulfate to specifically damage the neuromasts near the midline of zebrafish caused neutrophils to migrate out of the blood vessels and gather in the neuromasts. The number of neutrophils near the midline can be quantitatively observed ( Figure 3 ), calculate the inflammatory cell inhibition rate, inflammatory cell inhibition rate (%) = (the number of neutrophil migration in the inflammatory model group - the number of neutrophil migration in each experimental group) / the number of neutrophil migration in the inflammatory model group × 100%.

[0144] Table 2

[0145] Group Inflammatory cell inhibition rate (%) Example 1 30 Example 2 29 Example 3 28 Example 4 18 Example 5 20 Example 6 16 Example 7 23 Example 8 21 Example 9 20 Example 10 19 Comparative Example 1 0 Comparative Example 2 9 Comparative Example 3 9 Comparative Example 4 10 Positive control group 30

[0146] From the data in Table 2, it can be seen that the urea concentration of the dissolving solution used in Example 4 when purifying amelogenin exceeds 6M, and its inflammatory cell inhibition rate is lower than that of Example 1, indicating that too high urea will cause the purified product to contain more impurities, thereby affecting the anti-inflammatory activity of the amelogenin product;

[0147] The dissolving solutions of Example 6 and Comparative Example 1 do not contain TritonX-100 and urea respectively, and their inflammatory cell inhibition rates are lower than that of Example 1, indicating that TritonX-100 and urea can synergistically enhance the effect, which can not only improve the purity of amelogenin but also improve the anti-inflammatory activity of amelogenin.

[0148] The dissolving solutions of Examples 7 and 8 respectively contain one of β-mercaptoethanol and EDTA-2Na, and the inflammatory cell inhibition rates of both are lower than that of Example 1, indicating that the combined effect of β-mercaptoethanol and EDTA-2Na can effectively ensure the activity of amelogenin during the dissolution process, so that the prepared amelogenin product has stronger anti-inflammatory activity.

[0149] The refolding agent of Example 9 does not contain L-cysteine, and the refolding agent of Example 10 does not contain NaCl, glycerol, or EDTA-2Na. The inflammatory cell inhibition rates are both lower than those of Example 1, indicating that L-cysteine ​​can synergistically enhance the effect with NaCl, glycerol, and EDTA-2Na, and the obtained amelogenin product has stronger anti-inflammatory activity.

[0150] Compared with Example 1, the initial urea concentration of the dissolving solution of Example 5 is 1 M, and the inflammatory cell inhibition rate is lower than that of Example 1, indicating that when the initial urea concentration is 2 M, it is beneficial to enhance the activity of amelogenin.

[0151] Comparative Examples 2-4 respectively used a single concentration of urea solution and did not perform concentration gradient dissolution. Their inflammatory cell inhibition rates were lower than those of Example 1, indicating that the amelogenin prepared by the present invention using a urea concentration gradient solution has higher purity and activity.

[0152] Test Example 3

[0153] The repair effect of the amelogenin expressed in the form of inclusion bodies obtained in the examples and comparative examples was evaluated by the following specific method:

[0154] (1) Wild zebrafish that were 3 days post-fertilization and normally developed were randomly selected and anesthetized with 0.0002 g / mL tricaine. The tail fin was cut off and placed in a 28.5°C biochemical incubator for 2 h. Photos were taken under a stereomicroscope and then the zebrafish were transferred to a 96-well plate, with one zebrafish per well and 8 wells per group. The zebrafish were divided into an experimental group, a blank control group, and a positive control group.

[0155] 200 μL of the test substance was added to each well. The experimental groups were added with the amelogenin products prepared in the embodiments and comparative examples, the amelogenin product was a 0.006 g / L amelogenin solution prepared with zebrafish culture water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4·7H2O), the blank control group was added with zebrafish culture water; the positive control group was added with 0.006 g / L Rehmannia glutinosa solution (zebrafish culture water was used as solvent).

[0156] (2) Place in a 28.5°C biochemical incubator and incubate for 72 h. Take photos and record under a stereomicroscope. Figure 4 ), and the image analysis software Image J was used to count the area of ​​the zebrafish tail fin growth.

[0157] (3) The repair effect of the product was calculated by calculating the regenerated area of ​​the tail fin of the positive control group and the experimental group. The zebrafish tail fin repair promotion rate (%) = (the growth area of ​​the tail fin of each experimental group - the growth area of ​​the tail fin of the blank control group) / the growth area of ​​the tail fin of the blank control group × 100%.

[0158] Table 3

[0159]

[0160]

[0161] From the data in Table 3, it can be seen that the urea concentration of the dissolving solution used in Example 4 when purifying amelogenin exceeds 6M, and its zebrafish tail repair promotion rate is lower than that of Example 1, indicating that too high urea will cause the purified product to contain more impurities, thereby affecting the repair activity of the amelogenin product;

[0162] The dissolving solutions of Example 6 and Comparative Example 1 do not contain Triton X-100 and urea respectively, and their zebrafish tail repair promotion rates are lower than that of Example 1, indicating that Triton X-100 and urea can synergistically enhance the effectiveness, which can not only improve the purity of amelogenin but also improve the repair activity of amelogenin.

[0163] The dissolving solutions of Examples 7 and 8 respectively contain one of β-mercaptoethanol and EDTA-2Na, and the zebrafish tail repair promotion rates of both are lower than that of Example 1, indicating that the combined effect of β-mercaptoethanol and EDTA-2Na can effectively ensure the activity of amelogenin during the dissolution process, so that the obtained amelogenin product has stronger repair activity.

[0164] The refolding agent of Example 9 does not contain L-cysteine, and the refolding agent of Example 10 does not contain NaCl, glycerol, or EDTA-2Na. The zebrafish tail repair promotion rates are both lower than those of Example 1, indicating that L-cysteine ​​can synergistically enhance the effect with NaCl, glycerol, and EDTA-2Na, and the obtained amelogenin product has stronger repair activity.

[0165] Compared with Example 1, the initial urea concentration of the dissolving solution in Example 5 is 1 M, and the repair promotion rate of amelogenin is lower than that in Example 1, indicating that when the initial urea concentration is 2 M, it is beneficial to enhance the anti-inflammatory activity and repair activity of amelogenin.

[0166] Comparative Examples 2-4 respectively used a single concentration of urea solution and did not perform concentration gradient dissolution. Their amelogenin repair promotion rates were all lower than that of Example 1, indicating that the amelogenin repair activity obtained by purifying protein using a urea concentration gradient solution of the present invention is higher.

[0167] The applicant declares that the present invention illustrates the method and process of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0168] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0169] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing amelogenin expressed in the form of inclusion bodies, characterized in that: The preparation method comprises the following steps: (1) expressing a recombinant plasmid containing a human amelogenin gene in a prokaryotic expression system to obtain a recombinant human amelogenin in the form of inclusion bodies; (2) using a dissolving solution containing urea in a concentration gradient range of 2-6 M to dissolve the inclusion bodies in step (1) in a gradient manner, centrifuging to obtain a supernatant and a precipitate; (3) mixing the supernatant in step (2) with the refolding agent, filtering, and desalting the filtrate to obtain the human amelogenin expressed in the form of inclusion bodies; The components of the dissolving solution in step (2) also include 20 mM Tris buffer with a pH of 9.0, 1% TritonX-100, 1 mM β-mercaptoethanol and 0.1 mM EDTA-2Na; In step (2), the initial concentration of urea is 2 M, the sequential gradient dissolution is a gradient dissolution of urea concentration from low to high, the mass volume ratio of the inclusion body to the dissolving solution used for a single dissolution is 1:(8-15) g / mL, and the gradient is performed 2-5 times; The refolding agent in step (3) includes 20 mM Tris buffer, 200 mM NaCl, 10% glycerol by volume, 0.1 mM EDTA-2Na and 1 mM L-cysteine, and the pH of the refolding agent is 7-9.

2. The preparation method according to claim 1, characterized in that The prokaryotic expression system includes Escherichia coli, Bacillus subtilis or Lactobacillus.

3. The preparation method according to claim 1, characterized in that: The dissolving in step (2) is accompanied by shaking.

4. The preparation method according to claim 3, characterized in that: The shaking time is 1.5-3h.

5. The preparation method according to claim 1, characterized in that: The step (2) also includes a washing operation before the dissolution.

6. The preparation method according to claim 5, characterized in that: The washing solution used in the washing comprises Tris and / or TritonX-100.

7. The preparation method according to claim 6, characterized in that: The pH of the washing liquid is 7-10.

8. The preparation method according to claim 1, characterized in that: The centrifugal speed in step (2) is 8000-15000 rpm, and the centrifugal time is 15-45 min.

9. The preparation method according to claim 1, characterized in that: The specific operation of the mixing in step (3) is: dropping the supernatant into the refolding agent.

10. The preparation method according to claim 9, characterized in that: The process of dropping the supernatant into the refolding agent is accompanied by stirring.

11. The preparation method according to claim 1, characterized in that: The pore size of the filter membrane used in the filtration in step (3) is 0.45 μm.

12. The preparation method according to claim 1, characterized in that: The desalting column used in step (3) is a G25 desalting column.

13. Use of the preparation method according to any one of claims 1 to 12 in the preparation of oral care or beauty skin care products.

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