A method for preparing recombinant human nerve growth factor

Abstract

Provided is a method for preparing a recombinant human nerve growth factor, which can be used as a therapeutic drug.

Description

Technical Field

[0001] The invention belongs to the category of bioengineering and involves the preparation of high-purity, high-activity, unmodified recombinant human nerve growth factor by genetic engineering method. Background Art

[0002] Oxervate (cenegermin, recombinant human nerve growth factor, rhNGF) is an eye drop originally developed by Italian pharmaceutical company Dompé. It is an orphan drug for the treatment of adult patients with moderate to severe neurotrophic keratitis (NK) and is currently available in the EU and the U.S. In addition, its indication for the treatment of Alzheimer's disease is also under development.

[0003] In order to prepare recombinant human nerve growth factor, the original manufacturer first expressed proNGF inclusion bodies, then renatured, purified the proNGF renatured solution, and obtained recombinant human nerve growth factor through enzyme digestion and purification. However, this method requires enzyme digestion, and purification after enzyme digestion, with many steps and a cumbersome process.

[0004] Therefore, researchers have been hoping to find a simple method that can directly denature and renature. Collins, et al. (US5,986,070) invented a method for direct denaturation and renaturation of rhNGF, which uses Tris as a buffer, denatures under 8M urea conditions, and renatures under 8M urea conditions. This method obtains highly active rhNGF. However, the rhNGF obtained by this method was determined by mass spectrometry to have many impurities with molecular weights close to rhNGF. These impurities may be modified products of rhNGF. Since the molecular weight and properties of the modified rhNGF and the unmodified rhNGF are very close, this creates difficulties for subsequent separation and purification. Conventional methods are difficult to separate, and even if they are separated, the yield is low. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the present invention provides a method for preparing a high-purity, high-activity, unmodified recombinant human nerve growth factor. The inventors of the present application have found in their long-term research on rhNGF that when ammonium bicarbonate (NH 4 HCO 3 ) as a buffer, when renatured in the presence of urea, the recombinant human nerve growth factor prepared by the method of the present invention greatly reduces impurities of non-target proteins, has high purity, good biological activity, can be used as a therapeutic drug, and has good application prospects.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for preparing recombinant human nerve growth factor, comprising the following steps:

[0008] (a) expressing and isolating inclusion bodies of recombinant human nerve growth factor in Escherichia coli;

[0009] (b) washing the inclusion bodies using an inclusion body washing solution;

[0010] (c) adding a denaturing solution to dissolve the inclusion bodies;

[0011] (d) adding a renaturation solution to renature the inclusion bodies;

[0012] (e) purifying and obtaining recombinant human nerve growth factor;

[0013] Wherein, ammonium bicarbonate is used as a buffer in step (d).

[0014] The inclusion bodies of recombinant human nerve growth factor can be expressed and isolated using a commercial or known Escherichia coli protein expression system. According to a preferred embodiment of the present invention, the step (a) comprises the following steps: the amino acid sequence of human nerve growth factor is as shown in SEQ ID NO: 1, the human nerve growth factor gene is fully synthesized, inserted into a vector, transformed into Escherichia coli, expressed, broken into bacteria, centrifuged, and the precipitate is collected. According to a preferred embodiment of the present invention, the vector is pET28a, the Escherichia coli is the Escherichia coli expression strain BL21 (DE3) plyss, the expression is IPTG-induced expression, and the breaking of bacteria is ultrasonic breaking of bacteria or homogenizer breaking, etc.

[0015] After isolating the inclusion bodies of recombinant human nerve growth factor, the inclusion bodies can be washed with inclusion body washing solution to remove impurities. The inventors of the present application have made various attempts on the combination of inclusion body washing solutions to screen a washing scheme for the inclusion bodies of the recombinant human nerve growth factor that is particularly suitable for the present invention. According to a preferred embodiment of the present invention, the step (b) comprises the following steps: resuspending the precipitate with inclusion body washing solution A, ultrasonicating, centrifuging, and collecting the precipitate, wherein the inclusion body washing solution A comprises 20-100mM Tris, 50-150mM NaCl, 1-10mM EDTA, 0.5-3% Triton X-100, pH8.0-8.5; resuspending the precipitate with inclusion body washing solution B, ultrasonicating, centrifuging, and collecting the precipitate, wherein the inclusion body washing solution B comprises 20-100mM Tris, 50-150mM NaCl, 1-10mM EDTA, 1-4M urea, pH 8.0-8.5; and resuspending the precipitate with inclusion body washing solution C, ultrasonicating, centrifuging, and collecting the precipitate, wherein the inclusion body washing solution C comprises 20-100mM Tris, 50-150mM NaCl, 1-10mM EDTA, pH 8.0-8.5. More preferably, the step (b) comprises the following steps: resuspending the precipitate using inclusion body washing solution A, ultrasonicating, centrifuging, collecting the precipitate, and repeating the washing step once, wherein the inclusion body washing solution A comprises 50mM Tris, 100mM NaCl, 5mM EDTA, 1% Triton X-100, pH8.5; resuspending the precipitate using inclusion body washing solution B, ultrasonicating, centrifuging, collecting the precipitate, and repeating the washing step once, wherein the inclusion body washing solution B comprises 50mMTris, 100mM NaCl, 5mM EDTA, 2M urea, pH 8.5; and resuspending the precipitate using inclusion body washing solution C, ultrasonicating, centrifuging, collecting the precipitate, and repeating the washing step once, wherein the inclusion body washing solution C comprises 50mM Tris, 100mM NaCl, 5mM EDTA, pH 8.5.

[0016] After purifying the inclusion bodies of recombinant human nerve growth factor, the inclusion bodies need to be denatured and renatured to obtain the target protein with correct folding form and biological activity.

[0017] According to a preferred embodiment of the present invention, the step (c) comprises the following steps: adding a denaturing solution to the inclusion bodies, the denaturing solution comprising 8M urea and 15-25mM citric acid, pH 2.8-3.2, the inclusion bodies and the denaturing solution are added to the denaturing solution at a ratio of 1:20-30 in terms of wet weight (g): volume (ml), and the precipitate is removed by centrifugation after dissolution. According to a preferred embodiment of the present invention, the denaturing solution comprises 8M urea and 20mM citric acid, pH 3.0, the inclusion bodies and the denaturing solution are added to the denaturing solution at a ratio of 1:25 in terms of wet weight (g): volume (ml).

[0018] According to a preferred embodiment of the present invention, the step (d) comprises the following steps: adding 1 / 4 volume of 0.8-1.0M ammonium bicarbonate pH 8.0-9.0 and 8M urea solution to the denaturing solution, adding DTT to a final concentration of 5-10mM, 25-37°C for 30-60min; then adding oxidized glutathione to a final concentration of 20-40mM, 25-37°C for 10-15min; then adding 19 times volume of dilution buffer, the dilution buffer comprising 50-150mM Na 2 HPO 4 , 5-15mM ethanolamine, 4.2-4.6M urea, 14-18% PEG300, pH8.3-8.5; then add cysteine ​​to a final concentration of 1-5mM; drive out the air with argon gas, renature at 4°C for 1-7 days; use 3K membrane ultrafiltration to concentrate the renatured solution to a final rhNGF concentration of 1-2mg / ml. More preferably, the concentration of ammonium bicarbonate is 1M, the pH is 8.5, the final concentration of DTT is 5mM, the final concentration of oxidized glutathione is 20mM, and the dilution buffer includes 100mM Na 2 HPO 4 , 10 mM ethanolamine, 4.6 M urea, 15.8% PEG300, pH 8.3, the final concentration of cysteine ​​is 3 mM.

[0019] After the inclusion bodies are denatured and renatured, the target protein can be purified using a known chromatography system. According to one embodiment of the present invention, the step (e) comprises the steps of SP purification and C4 reverse phase chromatography.

[0020] Beneficial effects of the present invention:

[0021] 1. Simple operation, no need for enzyme cutting, fewer steps, less impurities in the obtained product and high purity;

[0022] 2. The method of the present invention does not destroy the biological activity of the target protein, and the obtained product can be used as a candidate drug.

[0023] The invention provides a method for preparing a recombinant human nerve growth factor, which has few impurities, high purity and good biological activity, can be used as a therapeutic drug and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the SDS-PAGE atlas of the expression product of the present invention, wherein M represents protein marker, US and UP represent the supernatant and precipitate of the strain without induced expression, and IS and IP represent the supernatant and precipitate of the strain induced expression by IPTG, respectively.

[0025] Figure 2 The SDS-PAGE map of the IPTG-induced expression product of the present invention, wherein M represents a protein marker.

[0026] Figure 3 This is the spectrum of the target protein of the present invention, wherein the molecular weight of rhNGF is 13261.65.

[0027] Figure 4 This is the spectrum of the target protein of the present invention, wherein the molecular weight of rhNGF is 13261.71.

[0028] Figure 5 is the target protein of the present invention promoting the proliferation of TF1 cells, wherein NGF represents the commercial rhNGF promoting the growth of TF1 cells, NH 4 HCO 3 represents the proliferation profile of TF1 cells promoted by rhNGF prepared by the method of the present invention, and Tris represents the proliferation profile of TF1 cells promoted by rhNGF prepared in Tris buffer. DETAILED DESCRIPTION

[0029] The following examples and experimental examples are provided to further illustrate the present invention, but are not intended to limit the present invention in any form.

[0030] Unless otherwise specified, the raw materials used in the following examples are all commercially available.

[0031] Example 1: Preparation of rhNGF inclusion bodies

[0032] 1.1 Construction of rhNGF expression strain

[0033] The two amino acids at the C-terminus of the mature peptide of human nerve growth factor NGF were removed, and the amino acid sequence was shown in SEQ ID NO:1. The codons were optimized according to the codon preference of Escherichia coli, and the human nerve growth factor gene was fully synthesized. Its gene sequence was shown in SEQ ID NO:2 and inserted into the pET28a multiple cloning site (MCS) XbaⅠ and XhoⅠ to construct the plasmid hNGFpET28a. The protein was expressed using the strong promoter T7. The plasmid hNGFpET28a was transformed into the Escherichia coli expression strain BL21(DE3)plyss (purchased from Promega) using 42 degrees heat shock, and the two colonies were induced for expression by IPTG, the bacteria were ultrasonically disrupted, and centrifuged at 12000rpm×5min. The SDS-PAGE patterns of the supernatant and precipitate of the expression product are shown below. Figure 1 The seed bacterial solution was added with 15% glycerol to prepare glycerol bacteria and stored at -80 degrees.

[0034] 1.2 IPTG-induced expression

[0035] The glycerol strain was inoculated with a resistance culture medium at a volume ratio of 1:1000, and cultured at 37 degrees and 180 rpm overnight; the seed liquid was inoculated with a resistance culture medium at a volume ratio of 1:100, and cultured at 37 degrees and 180 rpm until the OD600 was 0.3-0.8; IPTG was added to make the final concentration 1mM, and the culture was continued at 25 degrees and 170 rpm for 18 hours; centrifuged at 8500rpm×10min, the supernatant was removed, and the bacteria were collected; sampling was performed, the bacteria were broken by ultrasonication, and the precipitate was subjected to SDS-PAGE detection. The SDS-PAGE spectrum of the induced expression product is shown as follows Figure 2 shown.

[0036] 1.3 Isolation of inclusion bodies

[0037] The bacteria and the shredded bacteria solution (2mM Tris, 100mM NaCl, 5mM EDTA, pH 8.5) were added in a ratio of 1:5-1:10 of wet weight (g): volume (ml). Ultrasonication was performed for 2s with an interval of 3s at 80% power for 5min. The ultrasonic step was repeated four times. The solution was centrifuged at 12000rpm for 15min at 4℃ and the precipitate was collected.

[0038] 1.4 Purification of inclusion bodies

[0039] The inclusion bodies were resuspended in inclusion body washing solution A (50 mM Tris, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, pH 8.5), sonicated for 2 s and 3 s at intervals, power 80%, 5 min, the sonication step was repeated once, centrifuged at 12000 rpm × 15 min at 4°C, and the precipitate was collected. The washing step was repeated once.

[0040] The precipitate was resuspended in inclusion body washing solution B (50 mM Tris, 100 mM NaCl, 5 mM EDTA, 2 M urea, pH 8.5), sonicated for 2 s and 3 s at intervals, at 80% power for 5 min, the sonication step was repeated once, centrifuged at 12000 rpm for 15 min at 4°C, and the precipitate was collected. The washing step was repeated once.

[0041] The precipitate was resuspended in inclusion body washing solution C (50 mM Tris, 100 mM NaCl, 5 mM EDTA, pH 8.5), and sonicated for 2 s with an interval of 3 s at 80% power for 5 min. The sonication step was repeated once, and the precipitate was collected by centrifugation at 12000 rpm for 15 min at 4°C.

[0042] Example 2: Renaturation and purification of rhNGF inclusion bodies in ammonium bicarbonate

[0043] 2.1 Denaturation of inclusion bodies

[0044] The inclusion body precipitate in Example 1 and denaturation solution (8 M urea, 20 mM citric acid, pH 3.0) were added to the denaturation solution at a ratio of wet weight (g): volume (ml) of 1:25, and the precipitate was removed by centrifugation after dissolution.

[0045] 2.2 Renaturation of inclusion bodies

[0046] Add 1 / 4 volume of 1M ammonium bicarbonate pH 8.5 and 8M urea solution to the denaturing solution, add dithiothreitol (DTT) to a final concentration of 5mM, and incubate at 25°C for 30-60min; then add oxidized glutathione to a final concentration of 20mM, and incubate at 25°C for 10-15min; then add 19 times the volume of dilution buffer (100mM Na 2 HPO 4 , 10 mM ethanolamine, 4.6 M urea, 15.8% PEG300, pH 8.3); then add cysteine ​​to a final concentration of 3 mM; use argon to drive out the air and renature at 4 degrees for 1-7 days; use 3K membrane ultrafiltration to concentrate the renatured solution to a final rhNGF concentration of 1-2 mg / ml.

[0047] At the same time, a control sample was set up, the difference being that 1 / 4 volume of 1M Tris pH 8.5 and 8M urea were added to the denaturing solution, and other conditions were the same.

[0048] 2.3 Purification

[0049] 2.3.1 SP purification

[0050] SP column: SP XL 1ml GE

[0051] Loading: Buffer composition 20mM NaAc pH 5.0, conductivity 4.5

[0052] Elution conditions: gradient elution

[0053] A: 20 mM NaAc pH 5.0

[0054] B: 20 ​​mM NaAc 2 M NaCl pH 5.0

[0055] A to B 20 minutes.

[0056] 2.3.2 C4 reverse phase chromatography

[0057] Before loading, the protein was concentrated by ultrafiltration using a 3K membrane to a final rhNGF concentration of 0.8-1.2 / ml.

[0058] C4 column: Vydac 214TP 10μm C4 250×10mm

[0059] A: 0.1% trifluoroacetic acid H 2 O solution

[0060] B: 0.1% trifluoroacetic acid in acetonitrile

[0061] Chromatographic conditions: flow rate 5ml / min

[0062]

[0063] 2.4 Mass spectrometry detection

[0064] The mass spectrometry conditions were as follows:

[0065] Waters UPLC-XEVO G2Q-TOF LC-MS system. The system liquid phase part is configured as: BSM binary high pressure mixing pump, SM sample manager, TUV UV detector; the mass spectrometry part is configured as: ESI source, Q-TOF detector. Data processing and analysis are performed using Masslynx V4.1 and BiopharmaLynx analysis software (Version: 1.2).

[0066] liquid phase conditions

[0067] Chromatographic column: MassPREPTM Micro Desalting Column 2.1×5mm (intact protein molecular weight analysis), column temperature: 80°C;

[0068] Mobile phase A: 0.1% FA-H 2 O

[0069] Mobile phase B: 0.1% FA-CAN

[0070] Seal Wash Solution: 10% IPA

[0071] Mass spectrometer cleaning solution: 50% ACN

[0072] Mass Spectrometer IntelliStart Valve Cleaning Solution: 50% MeOH

[0073] Injection volume: 10 μL

[0074] Sample chamber temperature: 10°C

[0075] Gradient elution conditions:

[0076]

[0077] Mass spectrometry conditions

[0078] MS data were collected in the continuum mode under the resolution mode;

[0079] LockSpray acquisition mode: real-time acquisition and no calibration is applied.

[0080] Calibration solution: Real-time calibration (LockSpray) solution: 2 ng / μL LE solution;

[0081] Calibration solution for mass axis: 2 μg / μL sodium iodide solution.

[0082] Mass spectrometry parameters

[0083]

[0084] The mass spectra of rhNGF prepared using a control sample of Tris buffer were as follows Figure 3 As shown, there are many impurity peaks with molecular weights close to rhNGF. 4 HCO 3 Mass spectrometry of rhNGF prepared in buffer as Figure 4 As shown, there are almost no impurity peaks, which greatly improves the purity of the rhNGF sample.

[0085] Example 3: rhNGF biological activity assay

[0086] The rhNGF prepared in Example 2 and commercial rhNGF (purchased from Sino Biological) were tested for biological activity by TF1 cell proliferation assay.

[0087] The experimental steps are as follows:

[0088] 1. TF1 cells in logarithmic growth phase (ATCC@CRL-2003 TM) Wash twice with 1640 medium preheated at 37°C and centrifuge at 300-500 g for 5 min;

[0089] 2. Count TF1 cells, suspend them to an appropriate density with 1640 medium containing 10% FBS, and inoculate them into a 96-well plate at 10,000 cells / 150 μl / well;

[0090] 3. Dilute the rhNGF sample in a 96-well plate with 1640 medium, and dilute it three-fold for 9 times; add the diluted sample to the 96-well cell culture plate, 50 μl / well; fill the area around the 96 wells with 200 μl / well of distilled water;

[0091] 4. At 37°C, 5% CO 2 Incubate the culture in an incubator with appropriate conditions for 3 days (may be extended to 4 days depending on the situation);

[0092] 5. After 3 days, add 20 μl CCK-8 solution to each well of the 96-well cell culture plate and continue to culture in a 37°C incubator for 8 hours;

[0093] 6. After mixing, read the OD450 value with a microplate reader, and perform data analysis with GraphPad Prism6, draw graphs, and calculate EC50.

[0094] The results are as follows Figure 5 As shown, rhNGF prepared using Tris buffer and rhNGF prepared using the ammonium bicarbonate buffer of the present invention can cause TF1 cells to proliferate just like commercial rhNGF, indicating that rhNGF prepared by the method of the present invention has excellent biological activity and can be used as a candidate drug. Sequence Listing <110> Sunshine Guojian Pharmaceutical (Shanghai) Co., Ltd. <120> A preparation method of recombinant human nerve growth factor <130> SH363-20P450188 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 118 <212> PRT <213> Homo <400> 1 Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp 1 5 10 15 Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys 20 25 30 Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val 35 40 45 Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val 50 55 60 Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys 65 70 75 80 His Thr Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln 85 90 95 Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu 100 105 110 Ser Arg Lys Ala Val Arg 115 <210> 2 <211> 407 <212> DNA <213> Homo <400> 2 tctagaaata attttgttta actttaagaa ggagatatac catgagtagc agccatccga 60 ttttccatcg cggcgagttt agcgtgtgcg atagcgtgag cgttttgggtg ggcgataaaa 120 ccaccgccac cgacatcaaa ggcaggaag tgatggtgct gggcgaagtg atattaata 180 atagcgtgtt taaacagtac ttttttgaaa ccaaatgccg cgatccgaat ccggtggata 240 gcggttgccg cggcattgac agcaagcact ggaacagcta ctgcacaacc acccatacct 300 tcgtgaaagc tttaaccatg gatggcaaac aagctgcttg gcgctttatt cgtatcgata 360 ccgcttgtgt gtgcgtgctg agccgcaaag cagtgcgttg actcgag 407

Claims

1. A method for preparing recombinant human nerve growth factor, It is characterized in that The following steps are involved: (a) expressing and isolating inclusion bodies of recombinant human nerve growth factor in Escherichia coli; (b) washing the inclusion bodies using an inclusion body washing solution; (c) adding a denaturing solution to dissolve the inclusion bodies; (d) adding a renaturation solution to renature the inclusion bodies; (e) purifying and obtaining recombinant human nerve growth factor; in, The step (b) comprises the following steps: using inclusion body washing solution A to resuspend the precipitate, ultrasonicating, centrifuging, and collecting the precipitate, wherein the inclusion body washing solution A comprises 20-100mM Tris, 50-150mM NaCl, 1-10mM EDTA, 0.5-3% TritonX-100, and pH 8.0-8.5; using inclusion body washing solution B to resuspend the precipitate, ultrasonicating, centrifuging, and collecting the precipitate, wherein the inclusion body washing solution B comprises 20-100mM Tris, 50-150mM NaCl, 1-10mM EDTA, 1-4M urea, and pH 8.0-8.5; and then using inclusion body washing solution C to resuspend the precipitate, ultrasonicating, centrifuging, and collecting the precipitate, wherein the inclusion body washing solution C comprises 20-100mMTris, 50-150mM NaCl, 1-10mM EDTA, and pH 8.0-8.5; The step (c) comprises the following steps: adding a denaturing solution to the inclusion bodies, wherein the denaturing solution comprises 8M urea and 15-25mM citric acid, and has a pH of 2.8-3.2, adding the denaturing solution to the inclusion bodies at a ratio of 1:20-30 of wet weight (g): volume (ml), and centrifuging to remove the precipitate after dissolution; The step (d) comprises the following steps: adding 1 / 4 volume of 0.8-1.0M ammonium bicarbonate pH 8.0-9.0 and 8M urea solution to the denaturing solution, adding DTT to a final concentration of 5-10mM, 25-37°C for 30-60min; then adding oxidized glutathione to a final concentration of 20-40mM, 25-37°C for 10-15min; then adding 19 times volume of dilution buffer, the dilution buffer comprising 50-150mM Na 2 HPO 4 , 5-15mM ethanolamine, 4.2-4.6M urea, 14-18% PEG300, pH8.3-8.5; then add cysteine ​​to a final concentration of 1-5mM; use argon to drive out the air, and renature at 4°C for 1-7 days; use 3K membrane ultrafiltration to concentrate the renatured solution to a final rhNGF concentration of 1-2mg / ml.

2. The preparation method according to claim 1, It is characterized in that The step (a) comprises the following steps: the amino acid sequence of human nerve growth factor is as shown in SEQ ID NO: 1, the human nerve growth factor gene is fully synthesized, inserted into a vector, transformed into Escherichia coli, expressed, broken into bacteria, centrifuged, and precipitated.

3. The preparation method according to claim 2, It is characterized in that The vector is pET28a, the Escherichia coli is the Escherichia coli expression strain BL21 (DE3) plyss, the expression is IPTG-induced expression, and the bacteria are broken by ultrasonic or homogenizer.

4. The preparation method according to claim 1, It is characterized in that The step (b) comprises the following steps: using inclusion body washing solution A to resuspend the precipitate, ultrasonicate, centrifuge, collect the precipitate, and repeat the washing step once, wherein the inclusion body washing solution A comprises 50mM Tris, 100mM NaCl, 5mM EDTA, 1% Triton X-100, pH 8.5; using inclusion body washing solution B to resuspend the precipitate, ultrasonicate, centrifuge, collect the precipitate, and repeat the washing step once, wherein the inclusion body washing solution B comprises 50mMTris, 100mM NaCl, 5mM EDTA, 2M urea, pH 8.5; and then using inclusion body washing solution C to resuspend the precipitate, ultrasonicate, centrifuge, collect the precipitate, and repeat the washing step once, wherein the inclusion body washing solution C comprises 50mM Tris, 100mM NaCl, 5mM EDTA, pH 8.

5.

5. The preparation method according to claim 1, It is characterized in that The denaturing solution includes 8M urea and 20mM citric acid, pH 3.0, and the inclusion bodies are added to the denaturing solution at a ratio of wet weight (g): volume (ml) of 1:

25.

6. The preparation method according to claim 1, It is characterized in that The concentration of ammonium bicarbonate is 1 M, the pH is 8.5, the final concentration of DTT is 5 mM, the final concentration of oxidized glutathione is 20 mM, and the dilution buffer includes 100 mM Na 2 HPO 4 , 10 mM ethanolamine, 4.6 M urea, 15.8% PEG300, pH 8.3, the final concentration of cysteine ​​is 3 mM.

7. The preparation method according to claim 1, It is characterized in that The step (e) comprises the steps of SP purification and C4 reverse phase chromatography.

8. The preparation method according to claim 1, It is characterized in that The steps include: 1) expressing and isolating inclusion bodies of recombinant human nerve growth factor in Escherichia coli, wherein the amino acid sequence of the human nerve growth factor is as shown in SEQ ID NO: 1, the expression vector is pET28a, the Escherichia coli is the Escherichia coli expression strain BL21 (DE3) plyss, and the expression is IPTG-induced expression; 2) washing the inclusion bodies in step 1) respectively with an inclusion body washing solution, wherein: 2a) washing the inclusion bodies with inclusion body washing solution A, wherein the inclusion body washing solution A comprises 50 mM Tris, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, pH 8.5; 2b) washing the inclusion bodies obtained in step 2a) with inclusion body washing solution B, wherein the inclusion body washing solution B comprises 50 mM Tris, 100 mM NaCl, 5 mM EDTA, 2 M urea, pH 8.5; 2c) washing the inclusion bodies obtained in step 2b) with inclusion body washing solution C, wherein the inclusion body washing solution C comprises 50 mM Tris, 100 mM NaCl, 5 mM EDTA, pH 8.5; 3) adding a denaturing solution to dissolve the inclusion bodies obtained by washing in step 2), wherein the denaturing solution comprises 8M urea and 20mM citric acid, pH 3.0, and the inclusion bodies and the denaturing solution are added to the denaturing solution at a ratio of 1:25 in terms of wet weight (g): volume (ml); 4) renaturing the inclusion bodies obtained in step 3): adding 1 / 4 volume of 1.0M pH 8.5 ammonium bicarbonate and 8M urea solution to the denaturing solution in step 3), adding DTT to a final concentration of 5mM, and incubating at 25-37°C for 30-60min; then adding oxidized glutathione to a final concentration of 20mM, and incubating at 25-37°C for 10-15min; then adding 19 times volume of dilution buffer, wherein the dilution buffer includes 100mM Na 2 HPO 4 , 10 mM ethanolamine, 4.6 M urea, 15.8% PEG300, pH 8.3; then add cysteine ​​to a final concentration of 3 mM; use argon to drive out the air and renature at 4°C for 1-7 days; use 3K membrane ultrafiltration to concentrate the renatured solution to a final rhNGF concentration of 1-2 mg / ml.

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